Estriol therapy for autoimmune and neurodegenerative diseases and disorders

ABSTRACT

The present invention discloses administering steroid hormones to mammals to treat autoimmune related diseases, including post-partum auto immune diseases. Most preferably the invention uses estrogens, estranges, estriol or estrogen receptor active agents to prevent or ameliorate clinical symptoms of these Th1-mediated (cell-mediated) autoimmune diseases known to either have an initial onset following the birth of a child or which are exacerbated in patients in the post-partum period.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/722,672, filed Dec. 20, 2012, which is a continuation of U.S. patentapplication Ser. No. 11/992,558, filed Mar. 25, 2008, which claimspriority from International Patent Application No. PCT/US2006/037259,filed Sep. 26, 2006, which claims priority to U.S. ProvisionalApplication No. 60/833,527, filed Jul. 26, 2006, and U.S. ProvisionalApplication No. 60/720,971, filed Sep. 26, 2005, all of which areincorporated by reference herein.

U.S. patent application Ser. No. 11/992,558, to which this applicationclaims priority, is a continuation-in-part application of U.S. patentapplication Ser. No. 11/151,040, filed on Jun. 13, 2005, and issued asU.S. Pat. No. 8,372,826 on Feb. 12, 2013, which is a continuation ofapplication Ser. No. 10/131,834, filed on Apr. 24, 2002, and issued asU.S. Pat. No. 6,936,599 on Aug. 30, 2005, which claims priority to U.S.Provisional Application No. 60/286,842, filed on Apr. 25, 2001, all ofwhich are incorporated by reference herein.

This invention was made with Government support under Grant No. NS045443awarded by the National Institute of Neurological Disorders and Stroke,National Institutes of Health. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to steroidal therapies for treatingautoimmune diseases, and, more particularly, to administering primaryagents being estrogens or estrogen receptor active agents for thetreatment of cell mediated diseases. Optionally, secondary agents whicheffect the immune and/or nervous system may also be co-administered ortapered onto. This therapy may be used in patients, includingpost-partum patients. This invention also relates to steroidal therapiesfor the treatment of neurodegenerative diseases and disorders, includingcell mediated diseases. Finally, treatment kits are provided containingat least one primary agent and at least one secondary agent for treatinga patient presenting with symptomology of an autoimmune disease or aneurodegenerative disease or disorder.

2. General Background and State of the Art

There is a distinct female preponderance of autoimmune diseases duringthe reproductive ages including multiple sclerosis (MS), rheumatoidarthritis (RA), uveitis, myasthenia gravis (MG), Sjogren's syndrome, andHashimoto's thyroiditis.

For example, MS is a chronic, and often debilitating disease affectingthe central nervous system (brain and spinal cord). MS affects more than1 million people worldwide and is the most common neurological diseaseamong young adults, particularly woman. The exact cause of MS is stillunknown. MS attacks the nervous system resulting in myelin sheathssurrounding neuronal axons to be destroyed. This demyelinization cancause weakness, impaired vision, loss of balance, and poor musclecoordination. MS can have different patterns, sometimes leaving patientsrelatively well after episodes of acute worsening, sometimes leading toprogressive disability that persists after episodes of worsening. In theworst cases the disease can lead to paralysis or blindness.

Steroid hormones or sex-linked gene inheritance may be responsible forthe enhanced susceptibility of women to these autoimmune diseases. Arole for steroid hormones in susceptibility to autoimmune disease issupported by observations of alternations in disease symptomatology,with alterations in sex hormone levels such as during pregnancy,menopause or exogenous hormone administration (in the form of hormonereplacement (HRT) or oral contraceptives (ORC)). For example, women withMS and RA have been reported to experience remission of symptoms duringlate gestation. Particularly, MS patients have been reported to show adecrease in relapse rate in pregnancy.

Normally, cell-mediated immunity is mediated by T helper cell (Th1)secretion of interferon gamma (IFN-.gamma.) and tumor necrosis factorbeta (TNF-b). In contrast, humoral immunity is mediated by another groupof T helper cells (Th2) secreting interleukin (IL)-10, IL-4, IL-5 andIL-6. A systemic shift toward humoral immunity (or Th2-mediatedimmunity) has been noted during pregnancy. During pregnancy,cell-mediated immunity is decreased and humoral-mediated immunity isincreased thereby promoting fetal survival. Thus, this systemic shift inthe immune system may explain why cell-mediated diseases, including MSand RA have been reported to improve during pregnancy.

Although a shift toward humoral-mediated immunity has been demonstratedduring human pregnancy, mechanisms which induce this shift remainunclear. One possibility is local production of Th2 (or humoralmediated) cytokines by the placenta. Another possibility is theproduction of Th2 cytokines by immune cells, consequent to changedlevels of steroid hormones during pregnancy. Consistent with the latterpossibility, in vitro studies have demonstrated the ability of thesteroid progesterone to increase IL-4 production and the ability of thesteroid 17.beta.-estradiol to increase IL-10 production duringT-lymphocyte responses. However, it remains unclear what cellularmechanisms are involved in regulating in vivo amelioration of autoimmunesymptomology.

Examples of potential candidates which effect may effect MS duringpregnancy include: Sex hormones (estrogens, progesterone), cortisol,vitamin D, alpha-fetoprotein, human chorionic gonadotropin and pregnancyspecific glycoproteins.

Further, some studies have suggested that a unique pregnancy factortermed “early pregnancy factor” is responsible for improved progressionof cell-mediated autoimmune diseases during pregnancy. Other studieshave suggested a role for microchimerism. Still others suggest a rolefor local factors such as TGF.beta. or estriol (E3) which is known to beproduced by the placenta during pregnancy. Of note, E3 is at its highestserum levels in the third trimester of pregnancy. However, E3's role inameliorating symptoms of autoimmune diseases in humans is unclear.

Studies in laboratory animals have established that experimentalautoimmune encephalomyelitis (EAE) and other Th1 (cell-mediated)autoimmune diseases in mice improve during pregnancy.

Specifically, treatment with late pregnancy levels of estriol orsupraphysiological doses of estradiol (5 times pregnancy levels) wereshown to delay the onset of clinical EAE after disease wasexperimentally induced by immunization of mice (Jansson et al. 1994).However, there was no investigation as to how estrogens delayed the dayof onset of disease, nor as to whether disease severity was effected inthese animals once symptomology occurred.

In another study, it was shown that EAE disease severity could bereduced by treatment with estriol, either before or after disease onset.Treatment of EAE mice with 90 day release pellets of 5 milligrams or 15milligrams of estriol (E3) was shown not only to decrease diseaseseverity but also to enhance autoantigen specific humoral-immunity,increase production of the Th2 cytokine IL-10 and reduced inflammationand demyelination in EAE mice. Importantly, these changes in the diseasewere induced by a dose (5 mg) which was shown to yield estriol levels inserum that were similar to those which occur during late pregnancy (Kimet al., Neurology, 50(4 Supp. 4):A242-245, April 1998, FASEB Journal12(4):A616, March 1998 and Neurology 52(6):1230-1238, April 1999; hereinincorporated by reference). Thus, these results suggested that steroidhormones, and estriol in particular, may be involved in the ameliorationof autoimmune reactions in the EAE animal model.

Other groups later demonstrated that estrogen potentiated the effects oftreatment with TCR proteins to reduce autoimmune reactions in EAE mice.Offner, et al. FASEB Journal 14(6):A1246, April 2000; Int. Journal ofMol. Medicine 6 (Supp. 1): S8, October 2000 and Journal of Clin. Invest.105(10):1465-1472, May 2000). Further, it was shown in animal studiesthat estrogen suppressed the onset EAE in mice (Ito, et al. Journal ofImmunology, 167(1): 452-52, 2001) and that presumed diestrus levels ofestrogens reduced some manifestations of active EAE in mice. Bebo et al.Journal of Immunology 166(3): 2080-9, 2001.

However, the etiology and disease progression of EAE and MS are notidentical, thus it is unclear that estrogens alone would be effective inameliorating autoimmune responses in human patients. Indeed, not only isit unknown whether pregnancy doses of estrogens might be protective inhumans with autoimmune disease, it is unclear even in mice whether lowdoses of estrogens are protective. For example, it has been reported bysome that ovariectomy of female mice makes EAE disease worse (Matejuk etal., 2001), while others have found that ovariectomy had no effect ondisease severity (Kim et al., 2001; Voskuhl and Palaszynski, 2001a;Voskuhl and Palaszynski, 2001b). Thus, it is controversial whether lowlevels of estrogens, as they exist during the menstrual cycle, areprotective even in mice.

Data from human studies to date have shown no clear benefit of steroidsin treating any autoimmune disease. In humans, administration ofavailable hormone therapies (including HRTs and OCPs) containing amixture of sex hormones cause some autoimmune diseases to improve whileothers worsen.

For example, there has been no conclusive evidence that women areprotected from or have a decrease in symptomology or relapse rates dueto sex steroids. One study noted that past use of oral contraceptives inhealthy women had no effect on subsequent risk to develop MS (Hernan etal. 2000). Further, another study found that the incidence rates for MSin current users were not decreased as compared to never-users(Thorogood and Hannaford, 1998). Thus, low dose of the estrogens in oralcontraceptives are not of sufficient type or dose to ameliorate theimmunopathogenesis of MS even temporarily during intercurrent use. Atbest, in one study, patients had the subjective impression thatpre-existing MS symptoms (as opposed to relapse rates) worsen during thepremenstrual period and that the use of oral contraceptives may havedecreased this worsening (Zorgdrager and De Keyser, 1997). Importantly,the lack of reports of an effect of oral contraceptive therapy on MSrelapses is in marked contrast to what has been observed duringpregnancy.

In contrast, it has been shown that women had a lower the risk ofdeveloping MS during pregnancy compared to non-pregnant states(Runmarker and Andersen, 1995). Due to the numerous changes that occurduring pregnancy, hormonal and nonhormonal (as listed above), theetiology of the beneficial effect of pregnancy may or may be related tosex steroid fluctuations. It has also been reported for decades thatpregnancy decreases MS relapses (Abramsky, 1994; Birk et al. 1990; Birket al, 1998; Damek and Shuster, 1997; Runmarker and Andersen, 1995;Confavreux et al., 1998). These studies have shown that the latter partof pregnancy is associated with a significant reduction in relapses,while there is a rebound increase in relapses post partum. In contrast,the absence of such an effect on relapses during OCP or HRT indicatethat low level sex steroids are not adequate to treat these symptoms.

Further, women having rheumatoid arthritis that were treated with HRTdid not show significant improvement in their symptomology. DaSilva andHall, Baillieres Clinical Rheumatology 1992, 6:196-219; Bijlsma at al.Journal of Repro. Imm. 28(3-4):231-4, 1992; Hall et al. Annals of theRheumatic Diseases, 53(2): 112-6, 1994.

Thus, the low doses of hormones found naturally during the menstrualcycle or in ORT and HRT have not been shown to be effective atameliorating the symptomology of autoimmune diseases. This is in spiteof the observation that women having MS have a decreased relapse rateduring late pregnancy. Thus, a challenge has been to identify a hormoneand a treatment dose that is therapeutic in treating particularautoimmune diseases, while minimizing undesirable side effects.Obviously, the dose and method of administration of steroids in humansdiffers from steroid treatment in laboratory animals due to toxiceffects of prolonged exposure by patients to steroid hormones. Inparticular, there are clinical concerns of inducing breast orendometrial cancers in women requiring long term exposure to steroidhormones.

The actions of estrogen are mediated primarily by nuclear estrogenreceptors (ER) ER alpha and ER beta, although non-genomic membraneeffects have also been described previously. Originally it was thoughtthat ER alpha and ER beta would each have distinct tissue distributions,thereby providing a means through which use of selective estrogenreceptor modifiers. However, the relationship between ER alpha and ERbeat became complex, with most tissues expressing some detectable levelof each of these receptors. The two receptors at times did, and at othertimes did not, co-localize to the same cells within a given tissue.Furthermore, in some issues the two receptors were shown to actsynergistically, whereas in the other tissues they act antagonistically.However, any selective effects by ER alpha and ER beta on MS and otherauto-immune and Neurodegerative diseases have yet to be examined

Further, the direct and indirect neuroprotective mechanisms by estrogensin EAE are not necessarily mutually exclusive, and have yet to be fullyexplored. The finding that estrogens are neuroprotective in EAE,regardless of mechanism, has relevance to estrogen treatment in MS, aswell as pregnancy, a time when circulating estrogens are very high.Indeed, multiple pregnancies have been associated with a decrease inlong-term disability accumulation in MS (Runmarker and Andersen, 1995;Damek and Shuster, 1997). Because it is known that up to 5 years ofcontinuous treatment with immunomodulatory treatments are needed toimpact disability in MS, a temporary anti-inflammatory effect of thethird trimester of pregnancy would not necessarily be expected toimprove long-term disability. While the efficacy of estrogen treatmentappears to depend critically on its administration early, as apreventative therapy, before neurodegeneration has occurred (Mulnard etal., 2000), this therapeutic measure has yet to be explored.

Further, neurodegenerative diseases and disorders in addition to MScomprise a substantial clinical problem for which existing treatmentshave been ineffective at ameliorating the clinical symptomology orpreventing the progression of the disease or disorder.

Estrogen treatment has been shown previously to be neuroprotective in avariety of neurodegenerative disease models including Parkinson'sdisease, cerebellar ataxia, stroke, and spinal cord injury (Leranth etal., 2000; Dubal et al., 2001; Wise et al., 2001; Jover et al., 2002;Rau et al., 2003; Sierra et al., 2003; Sribnick et al., 2003, 2005).Estrogens are lipophilic, readily traversing the blood-brain barrier,with the potential to be directly neuroprotective (Brinton, 2001;Garcia-Segura et al., 2001; Wise et al., 2001). Estrogen-mediatedprotection of neurons has been demonstrated in a variety of in vitromodels of neurodegeneration including those induced by excitotoxicityand oxidative stress (Behl et al., 1995; Goodman et al., 1996; Behl etal., 1997; Harms et al., 2001). Estrogens have also been shown todecrease glutamate-induced apoptosis and preserve electrophysiologicfunction in primary cortical neurons (Sribnick et al., 2003, 2004). Inaddition, in vitro studies have demonstrated the ability of estrogen tomodulate the astrocytic response to injury (Azcoitia et al., 1999;Garcia-Segura et al., 1999) and protect oligodendrocytes fromcytotoxicity (Sur et al., 2003; Cantarella et al., 2004; Takao et al.,2004). However, the role of estrogen and estrogen receptor subtypesinvolved neuroprotection has yet to be fully explored.

INVENTION SUMMARY

A general object of the present invention is to provide a method ofadministering steroid hormones to mammals to treat autoimmune relateddiseases, more particularly, Th1-mediated (cell-mediated) autoimmunediseases including: multiple sclerosis (MS), rheumatoid arthritis (RA),autoimmune thyroiditis, uveitis and other autoimmune diseases in whichclinical symptomology has shown improvement during the third term ofpregnancy. The method may also include the treatment of post-partumpatients having been diagnosed with, or at risk for developingautoimmune diseases, including MS. The method may also include thetreatment of patients having been diagnosed with, or at risk fordeveloping neurodegenerative diseases, including MS.

In accordance with one aspect of the present invention, these objectivesare accomplished by providing a treatment for autoimmune relateddiseases with a selected dose and course of a primary agent being anestrogen or estrogen receptor-effective composition. The primary agentmay include estrogen receptor selective ligands, such as agonists whichmimic the effect of estrogens.

In accordance with one aspect of the present invention, these objectivesare accomplished by providing a patient with a therapeutically effectiveamount of estriol, comprising from about 4 to 16 milligrams per day, ormore specifically, about 8 milligrams once daily via oraladministration.

In accordance with another aspect of the present invention, theseobjectives are accomplished by providing a therapeutically effectiveamount of a primary agent in combination with a therapeuticallyeffective amount of a secondary active agent, such as progesterone,glucocorticoids and/or known or experimental drugs used to treatautoimmune diseases.

In accordance with one aspect of the present invention, the inventioncomprises the use of a primary agent comprising an estrogen receptoralpha ligand having anti-inflammatory and/or neuroprotective effects toprevent or ameliorate clinical symptoms of autoimmune and/orneurodegenerative diseases or disorders, including multiple sclerosis.

In accordance with one aspect of the present invention, the inventioncomprises the use of a primary agent comprising an estrogen receptorbeta ligand having neuroprotective effects to prevent or ameliorateclinical symptoms of neurodegenerative diseases or disorders, includingmultiple sclerosis.

The above described and many other features and attendant advantages ofthe present invention will become apparent from a consideration of thefollowing detailed description when considered in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic depicting the trial design described in Example1; FIG. 1B is a bar graph depicting human serum levels during pregnancy,estriol treatment (Tx), and pretreatment (Pre Tx levels).

FIG. 2A is a bar graph describing the Delayed Type Hypersensitivity(DTH) responses to tetanus and to candida; FIG. 2B is a bar graphdepicting levels of IFN.gamma. between treatment groups.

FIGS. 3A-F are bar graphs depicting each patient's gadolinium enhancinglesion volumes on serial cerebral MRIs which were assessed at each monthduring the pretreatment, estriol treatment and post treatment periods.

FIG. 4 is a bar graph depicting mean percent change in PASAT scoresduring treatment with estriol as compared to pretreatment.

FIGS. 5A-C are bar graphs showing the uterine weights of wild type (WT),ER beta knock-out (KO), or ER alpha KO in mice treated with a control(vehicle), estrogen receptor alpha ligand (PPT) or estradiol treatedanimals (y-axis=uterine weight in grams).

FIGS. 6A-C are graphs showing the effect of ER alpha selective ligand onclinical scores in wild type (WT), ER beta knock-out (KO), or ER alphaKO in mice treated with a control (vehicle), estrogen receptor alphaligand (PPT) or estradiol treated animals.

FIGS. 7A-D are bar graphs showing proinflammatory cytokine production byperipheral immune cells in ovariectomized, wild type (WT) C57BL/6 femalemice with EAE.

FIGS. 8A-E depict various measures of estrogen receptor alpha ligandreduced inflammation and demyelination in spinal cords of mice with EAE.FIG. 8A are thoracic spinal cord sections from normal, or treated mice(vehicle, estradiol (E2) or estrogen receptor alpha ligand (PPT)); FIG.8B depicts luxol fast-blue stained magnified regions of the dorsalspinal column for the same sections as shown in 8A (40× magnification);FIG. 8C depicts anti-BMP immunostained magnified regions of the dorsalspinal column for the same sections as shown in 8A; FIG. 8D is a bargraph showing white matter cell density by treatment group; and FIG. 8Eis a bar graph showing myelin density by treatment group.

FIGS. 9A-E depict various measures of estrogen receptor alpha ligandreduced inflammation and demyelination in spinal cords of mice with EAE.FIGS. 9A-D are split images of thoracic spinal cord sections stainedwith NeuN⁺ (red) in I and Niss1 in ii at 4× magnification, derived frommice from each treatment group (normal, vehicle, estradiol (E2) orestrogen receptor alpha ligand (PPT)). FIG. 9E is a bar graph showingthe number of NeuN⁺ immunolabeled neurons in the delineated gray matter.

FIGS. 10A-D depict various measures of estrogen receptor alpha ligandreduced inflammation and demylination in spinal cords of mice with EAE.FIGS. 10A and B are images of thoracic spinal cord sections shown inFIG. 5 co-immunostained with NF200 (green) and CD45 (red) at 10×magnification, derived from mice from each treatment group (normal,vehicle, estradiol (E2) or estrogen receptor alpha ligand (PPT)). FIG.10C is a bar graph showing the axon number and FIG. 10D is a bar graphshowing Mac-3 cell density measurements.

FIG. 11 is a bar graph showing the uterine weights of wild type (WT),estrogen receptor alpha ligand (PPT) and estrogen receptor beta ligand(DPN) treated animals (y-axis=uterine weight in grams).

FIGS. 12A-G are graphs showing the effect on clinical scores of wildtype (WT), estrogen receptor alpha ligand (PPT) and estrogen receptorbeta ligand (DPN) treated animals.

FIGS. 13A-C are bar graphs showing the effect of treatment with aestrogen receptor selective ligand (DPN), vehicle or estradiol onproliferation or cytokine production.

FIGS. 14A-F depict various measures of estrogen receptor alpha ligandreduced inflammation and demyelination in spinal cords of mice with EAE.FIGS. 14A and 14C are early and late thoracic spinal cord sections fromnormal, or treated mice (vehicle, estrogen receptor alpha (PPT) orestrogen receptor beta ligand (DPN)); FIG. 14B depicts early whitematter cell density for each treatment group; FIG. 14D depicts latewhite matter cell density for each treatment group; 14 E and F depictearly and late sections co-immunostained with NF200 (green) and CD45(red) at 10× magnification, derived from mice from each treatment group.

FIGS. 15A-H depict various measures of estrogen receptor alpha and betaligand preservation of MBP and spare axonal pathology in spinal cords ofEAE mice. FIGS. 15A and 15C are images of thoracic spinal cord sectionsstained with NeuN (red) 10× magnification, derived from mice at earlyand late time points from each treatment group (normal, vehicle,estrogen alpha ligand (PPT) or estrogen receptor beat ligand (DPN)).FIGS. 15E and 15G are images of thoracic spinal cord sectionsco-immunostained with anti-NF200 (green, i) and anti-BMP (red, ii),shown merged in iii, derived from mice at early and late time pointsfrom each treatment group (normal, vehicle, estrogen alpha ligand (PPT)or estrogen receptor beat ligand (DPN)); FIGS. 15B and 15D are bargraphs showing myelin density, early and late, respectively, while FIGS.15F and 15H show axon number, early and late, respectively.

FIGS. 16A-C depict various measures of estrogen receptor alpha and betaligand preservation of neuronal staining of gray matter in spinal cordsof mice with EAE. FIGS. 16B and 16D are bar graphs showingquantification of NeuN+ cells in the gray matter.

FIGS. 17A-B are images of thoracic spinal cord sections stained derivedfrom ERβ knock out control mice, vehicle-treated mice with EAE, andDPN-treated mice with EAE. FIGS. 17C-F depict quantification of whitematter cell density, myelin density, axonal numbers and NeuN+ cells incontrol mice, vehicle-treated mice with EAE, and DPN-treated mice withEAE.

FIGS. 18A-B depict results in recovery of motor function during EAE incontrol, vehicle-treated, and DPN-treated mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This description is not to be taken in a limiting sense, but is mademerely for the purpose of illustrating the general principles of theinvention. The section titles and overall organization of the presentdetailed description are for the purpose of convenience only and are notintended to limit the present invention.

Generally, the invention involves a method of treating mammal exhibitingclinical symptoms of an autoimmune disease comprising administering aprimary agent at a therapeutically effective dosage in an effectivedosage form at a selected interval. The treatment is aimed at reducingthe symptomology and/or progression of the disease. In the preferredembodiment of the invention, human patients clinically diagnosed with MS(including both relapsing remitting or secondary progressive typepatients) are treated with an oral preparation of 8 milligrams estrioldaily and have ameliorated symptomology.

Amelioration of the autoimmune disease refers to any observablebeneficial effect of the treatment. The beneficial effect can beevidenced by a delayed onset or progression of disease symptomology, areduction in the severity of some or all of the clinical symptoms, or animprovement in the overall health.

For example, patients who have clinical symptoms of an autoimmunedisease often suffer from some or all of the following symptoms:worsening of pre-existing symptoms (such as joint pain in rheumatoidarthritis), the appearance of new symptoms (new joints affected inrheumatoid arthritis) or increased generalized weakness and fatigue. MSpatients in particular suffer from the following symptoms: weakness,numbness, tingling, loss of vision, memory difficulty and extremefatigue. Thus an amelioration of disease in MS would include a reductionin the frequency or severity of onset of weakness, numbness, tingling,loss of vision, memory difficulty and extreme fatigue. On imaging of thebrain (MRI) amelioration of disease would be evidenced by a decrease inthe number or volume of gadolinium enhancing lesions, a stabilization orslowing of the accumulation of T2 lesions and/or a slowing in the rateof atrophy formation. Immunologically, an increase in Th2 cytokines(such as IL-10) a decrease in Th1 cytokines (such as interferon gamma)would be associated with disease amelioration.

Patients may also express criteria indicating they are at risk fordeveloping autoimmune diseases. These patients may be preventativelytreated to delay the onset of clinical symptomology. More specifically,patients who present initially with clinically isolated syndromes (CIS)may be treated using the treatment paradigm outlined in this invention.These patients have had at least one clinical event consistent with MS,but have not met full criteria for MS diagnosis since the definitediagnosis requires more than one clinical event at another time(McDonald et al., 2001). Treatment of the present invention would beadvantageous at least in preventing or delaying the development ofclinically definite MS.

PRIMARY AGENT. The primary agent useful in this invention is a steroidhormone, more particularly a estrogen or a steroidal or non-steroidalestrogen receptor active agent. Most preferably the primary agent isestriol (estra-1,3,5(10)-triene-3,16,17-triol), E3, such as estriolsuccinate, estriol dihexanate or estriol sulfmate. However, the primaryagent may be precursors or analogs of estriol (such as nyestriol),estrone (E1) or precursors or analogs of estrone, 17.beta.-estradiol(E2) or precursors (including aromatizable testosterone) or analogs of17.beta.-estradiol, or estranges.

The primary agent may also be a metabolite or derivatives of E1, E2 orE3 which are active at the estrogen receptor .alpha. or .beta.Metabolites and derivatives may have a similar core structure to E1, E2or E3 but may have one or more different groups (ex. hydroxyl, ketone,halide, etc.) at one or more ring positions. Synthetic steroids whichare effective at estrogen receptor are also useful in this invention,such as those described in WO 97/08188 or U.S. Pat. No. 6,043,236 toBrattsand, which is hereby incorporated by reference herein.

The primary agent may also be an estrogen receptor .alpha. or .beta.,agonists and/or antagonist. These agonists or antagonists may besteroidal or non-steroidal agents which bind to and/or cause a change inactivity or binding of at least one of the estrogen receptor .alpha. or.beta. subtypes. For example, specific agonists of ER alpha and ER betamay be useful in this invention (Fritzmeier, et al.). Doses of theseagonists may be titrated to achieve an effect on disease similar to thatwhich is observed during pregnancy and during treatment with pregnancydoses of estriol by methodologies known to those skilled in the art ofsteroid pharmacology.

Any one or combination of these estrogens or estrogen receptor activeagents may be used to treat the selected autoimmune disease. Theselection of the estrogens or estrogen receptor active agents can bemade considering secondary side effects of the treatment to the patient.For example, estriol may be selected over 17.beta.-estradiol, becauseestriol causes minimal endometrial proliferation and is not associatedwith increased risk of breast cancer.

Minimal endometrial proliferation is observed when the long-actingestriol derivative, nyestriol is used. Indeed, because estriol haspartial antagonist action on the binding of 17.beta.-estradiol to theestrogen receptor in vivo, estriol was at one point in the pastconsidered as a therapeutic agent for treatment and prevention of breastcancer.

THERAPEUTICALLY EFFECTIVE DOSAGE OF THE PRIMARY AGENT. A therapeuticallyeffective dose of the primary agent is one sufficient to raise the serumconcentration above basal levels, and preferably to pregnancy levels orabove pregnancy levels. Most preferably, the therapeutically effectivedosage of the primary agent is selected to result in serum levels in apatient equivalent to the steroid hormone level of that agent in womenin the second or third trimester of pregnancy.

For example, during the normal female menstrual cycle estradiol levelsare in the range of about 350 pg/ml serum. During pregnancy, there isabout a 100 fold increase in the level of estradiol to about 10,000 toabout 35,000 pg/ml serum. (Correale, et al. Journal of Immunology161:3365 (1998) and Gilmore, et al. Journal of Immunology 158:446). Incontrast, estriol levels are undetectable during the menstrual cycle inthe non-pregnant state. Estradiol levels rise progressively duringpregnancy to levels from 3,000 to 30,000 pg/ml (3 to 30 ng/ml)(www.il-st-acad-sci.org/steroid 1.html#se3t).

In one embodiment, where the primary agent is estriol, the preferabledose is from about 4 to 16 milligrams daily, and more specifically,about 8 milligrams daily. In this embodiment, blood serum levelspreferably reach at least about 2 ng/ml, may reach about 10 to about 35ng/ml, or most preferably about 20-30 ng/ml. (Sicotte et al. Neurology56:A75). In some embodiments, estradiol (E2) levels would preferablyreach at least about 2 ng/ml and most preferably about to 10-35 ng/ml.In some embodiments, estrone (E1) levels would preferably reach at leastabout 2 ng/ml and most preferably about 5-18 ng/ml (DeGroot and Jameson,1994).

The dosage of the primary agent may be selected for an individualpatient depending upon the route of administration, severity of disease,age and weight of the patient, other medications the patient is takingand other factors normally considered by the attending physician, whendetermining the individual regimen and dosage level as the mostappropriate for a particular patient.

The use of this group of primary agents is advantageous in at least thatother known or experimental treatments for cellular mediated autoimmunediseases are chemotherapeutic immunosuppressants which have significantrisks and side effects to patients, including decreasing the ability ofthe patient to fight infections, inducing liver or heart toxicity whichare not caused by estrogen treatment. Other agents used in MS do notcause these side effect, but are associated with flu-like symptoms orchest tightness. Further, these previously used agents are associatedwith local skin reactions since they entail injections at frequenciesranging from daily to once per week.

DOSAGE FORM. The therapeutically effective dose of the primary agentincluded in the dosage form is selected at least by considering the typeof primary agent selected and the mode of administration. The dosageform may include the active primary agent in combination with otherinert ingredients, including adjutants and pharmaceutically acceptablecarriers for the facilitation of dosage to the patient as known to thoseskilled in the pharmaceutical arts. The dosage form may be any formsuitable to cause the primary agent to enter into the tissues of thepatient.

In one embodiment, the dosage form of the primary agent is an oralpreparation (liquid, tablet, capsule, caplet or the like) which whenconsumed results in elevated serum estrogen levels. The oral preparationmay comprise conventional carriers including dilutents, binders, timerelease agents, lubricants and disinigrants.

In other embodiments of the invention, the dosage form may be providedin a topical preparation (lotion, creme ointment or the like) fortransdermal application. Alternatively, the dosage form may be providedin a suppository or the like for transvaginal or transrectalapplication.

That estrogens or estrogen receptor active agents can be delivered viathese dosage forms is advantageous in that currently availabletherapies, for MS for example, are all injectables which areinconvenient for the user and lead to decreased patient compliance withthe treatment. Non-injectable dosage forms are further advantageous overcurrent injectable treatments which often cause side effects in patientsincluding flu-like symptoms (particularly, .beta. interferon) andinjection site reactions which may lead to lipotrophy (particularly,glatiramer acetate copolymer-1).

However, in additional embodiments, the dosage form may also allow forpreparations to be applied subcutaneously, intravenously,intramuscularly or via the respiratory system.

SECONDARY ACTIVE AGENTS. Any one or a combination of secondary activeagents may be included in the dosage form with the primary agent.Alternatively, any one or a combination of secondary active agents maybe administered independently of the primary agent, but concurrent intime such that the patient is exposed to at least two agents for thetreatment of their immunological disease.

The secondary agents are preferably immunotherapeutic agents, which actsynergistically with the primary agent to diminish the symptomology ofthe autoimmune disease. Secondary active agents may be selected toenhance the effect of the estrogen or estrogen receptor active agent,reduce the effect of the estrogen or estrogen receptor active agent oreffect a different system than that effected by the estrogen or estrogenreceptor active agent.

Secondary active agents include immunotherapeutic agents which cause achange in the activity or function of the immune system.

In one embodiment, a secondary agent may be a therapeutically effectiveamount of progesterone, precursor, analog or progesterone receptoragonist or antagonist. Most preferably, the secondary agent is 100-200milligrams of progesterone administered daily. Progesterone incombination with estrogen or estrogen receptor active agent treatment isadvantageous in at least protecting patients against risks associatedwith long term estrogen exposure, including, but not limited toendometrial proliferation and breast cancers.

In another embodiment, a secondary agent may be a therapeuticallyeffective amount of glucocorticoid, precursor, analog or glucocorticoidreceptor agonist or antagonist. For example, prednisone may beadministered, most preferably in the dosage range of about 5-60milligrams per day. Also, methyl prednisone (Solumedrol) may beadministered, most preferably in the dosage range of about 1-2milligrams per day. Glucocorticoids are currently used to treat relapseepisodes in MS patients, and symptomatic RA within this dosage range.

In other embodiments, a secondary agent may be selected from the groupimmunotherapeutic compounds. For example, as .beta.-interferon (Avonex®(interferon-beta 1a), Rebiff® (by Serono); Biogen, Betaseron®(interferon-beta 1b) Berlex, Schering), glatiramer acetate copolymer-1(Copaxone®; Teva), antineoplastics (such as mitoxantrone; Novatrone®Lederle Labs), human monoclonal antibodies (such as natalizumab;Antegren® Elan Corp. and Biogen Inc.), immonusuppressants (such asmycophenolate mofetil; CellCept® Hoffman-LaRoche Inc.), paclitaxel(Taxol®; Bristol-Meyers Oncology), cyclosporine (such as cyclosporin A),corticosteroids (glucocorticoids, such as prednisone and methylprednisone), azathioprine, cyclophosphamide, methotrexate, cladribine,4-aminopyridine and tizanidine and natalizumab (Tysabri)

By way of example, which is consistent with the current therapeutic usesfor these treatments, Avonex® in a dosage of about 0 to about 30 mcg maybe injected intramuscularly once a week. Betaseron® in a dosage of about0 to about 0.25 mg may be injected subcutaneously every other day.Copaxone® in a dosage of about 0 to about 20 mg may be injectedsubcutaneously every day. Finally, Rebiff® may be injected at atherapeutic dose and at an interval to be determined based on clinicaltrial data. Further, any of these secondary agents may be used inincreasing, constant or decreasing dose in combination with a primaryagent, such as estriol or an ER alpha or beta receptor ligand. However,dosages and method of administration may be altered to maximize theeffect of these therapies in conjunction with estrogen treatment.Dosages may be altered using criteria that are known to those skilled inthe art of diagnosing and treating autoimmune diseases.

Preferably, secondary agents would be administered in the dosage rangescurrently used to treat patients having autoimmune diseases, includingMS patients. Alternatively, the secondary agents may be administered ata reduced dose or with reduced frequency due to synergistic orduplicative physiological effects with the primary agent.

Preferably, patients exhibiting symptomology of autoimmune diseases aretreated with the above agents (estrogen or estrogen receptor activeagents with or without secondary agents). Most preferably, patientsexhibit autoimmune diseases marked by improvement in symptomology atleast during a treatment regimen, including but not limited to thatreflecting patterns observed during the second or third trimester ofpregnancy.

Treatment of Post-Partum Patients.

In a recent clinical study, a dramatic decrease in the relapse rateduring pregnancy, especially in the third trimester was noted, with arebound increase in the three months post partum (such as a patient whohas given birth, including until the following year from the date ofbirth). These data, in addition to confirmatory animal testing using theEAE model suggest that sex steroids have profound effects in autoimmunedisease progression and symptomology, and could also have an effect onmyelinating and re-myelinating the peripheral and possibly the centralnervous system.

In another embodiment of the invention, the invention may includemethods of steroidal therapies for preventing or treating femalepost-partum patients, expressing symptoms of or at risk for autoimmunediseases. The invention may include the method of preventing or treatinga subject having been diagnosed with at least one symptom of anautoimmune disease to reduce the symptomology of/and or slow theprogression of the disease. The method according to the invention maycomprise administering primary agents being estrogens or estrogenreceptor active agents for the treatment of cell mediated diseases. Theinvention may further include the treatment with secondary agents whicheffect the immune system, which may be co-administered or tapered onto.In other embodiments, the use of the primary agents, combinations ofprimary agents with secondary agents, at the doses and in the dosageforms may be administered as described above for auto immune diseases.

In one embodiment of the invention, human post-partum patients who areclinically diagnosed with an autoimmune disease, such as MS (includingboth relapsing remitting or secondary progressive type patients) may betreated with an oral preparation of 8 milligrams estriol daily,resulting in ameliorated symptomology. Additionally, patients could beadministered an estriol or an estrogen following birth, then taperedonto a conventional FDA approved therapy, such as Copaxone.

Amelioration of the post-partum autoimmune disease refers to anyobservable beneficial effect of the treatment. The beneficial effect canbe evidenced by a delayed onset or progression of disease symptomology,a reduction in the severity of some or all of the clinical symptoms, oran improvement in the overall health.

For example, patients who have clinical symptoms of an autoimmunedisease often suffer from some or all of the following symptoms:worsening of pre-existing symptoms (such as joint pain in rheumatoidarthritis), the appearance of new symptoms (new joints affected inrheumatoid arthritis) or increased generalized weakness and fatigue.Multiple sclerosis patients in particular suffer from the followingsymptoms: weakness, numbness, tingling, loss of vision, memorydifficulty and extreme fatigue. Thus an amelioration of disease inmultiple sclerosis would include a reduction in the frequency orseverity of onset of weakness, numbness, tingling, loss of vision,memory difficulty and extreme fatigue. On imaging of the brain (MRI)amelioration of disease would be evidenced by a decrease in the numberor volume of gadolinium enhancing lesions, a stabilization or slowing ofthe accumulation of T2 lesions and/or a slowing in the rate of atrophyformation. Immunologically, an increase in Th2 cytokines (such as IL-10)a decrease in Th1 cytokines (such as interferon gamma) would beassociated with disease amelioration.

Patients may also express criteria indicating they are at risk fordeveloping autoimmune diseases. These patients may be preventativelytreated to delay the onset of clinical symptomology. More specifically,patients who present initially with clinically isolated syndromes (CIS)may be treated using the treatment paradigm outlined in this invention.These patients have had at least one clinical event consistent with MS,but have not met full criteria for MS diagnosis since the definitediagnosis requires more than one clinical event at another time(McDonald et al., 2001). Treatment of the present invention would beadvantageous at least in preventing or delaying the development ofclinically definite MS.

Treatment with Primary Agents being ER Alpha Receptor Agonists.

In one embodiment, the invention comprises the use of a primary agentcomprising an estrogen receptor alpha ligand, such as an agonist, havingan anti-inflammatory and neuroprotective effect to prevent or ameliorateclinical symptoms of auto immune diseases including multiple sclerosis.

As above, multiple sclerosis is an inflammatory, neurodegenerativedisease for which experimental autoimmune encephalomyelitis (EAE) is amodel. Treatments with estrogens have been shown to decrease theseverity of EAE through anti-inflammatory and neuropreservationmechanisms. More recently, it has been determined that estrogen receptoralpha (ER alpha) ligand could recapitulate the estrogen-mediatedprotection in clinical EAE. As described in the examples below, EAEtreatment with a highly selective ER alpha agonist (propyl pyrazoletriol) ameliorated clinical disease in both wild-type and ER betaknock-out mice, but not in ER alpha knock-out mice, suggesting that theER alpha ligand maintained ER alpha selectivity in vivo during disease.Anti-inflammatory and neuroprotective effects included, reducedauto-antigen-specific pro-inflammatory cytokine production, increasedanti-inflammatory cytokines, reduced nervous system inflammation,reduced demyelination, reduction in neuronal cell loss, reduction inaxonal transaction, decreased white matter lesions, decreased loss inaxonal number, reduced nervous system monocyte activation and reducednervous system microglial activation. See Examples 5 and 6 and FIGS.5-10.

Treatment of Patients with Neurodegenerative Diseases/Disorders.

In another embodiment of the invention, the invention comprises thetreatment of neurodegenerative diseases and disorders, including MS. Theinvention may include the method of preventing or treating a subjecthaving been diagnosed or exhibiting at least one clinical symptom of aneurodegenerative disease or disorder.

The method according to the invention may comprise administering aprimary agent at a therapeutically effective dosage in an effectivedosage form at a selected interval to prevent, reduce the frequency orreduce the severity of the symptoms and/or progression of the disease ordisorder.

In one specific embodiment, the method may comprise administration of 8milligrams estriol daily, such as in an oral preparation and result inameliorated symptomology. In one other embodiment, the method maycomprise treating the patent with a combination of estrogen andprogestin or progesterone, as a secondary agent. In other embodiments,the use of the primary agents, combinations of primary agents withsecondary agents, at the doses and in the dosage forms may beadministered as described above for auto immune diseases.

In other embodiments, the primary agent may comprise an estrogenreceptor beta ligand, such as a estrogen receptor beta agonist. In theEAE animal model, an estrogen receptor beta agonist was found to havesignificant neuroprotective effects, including reduced demyelination,reduces axon loss, reduces neuronal abnormalities and reduced motorimpairment, and reduced relapses. See Example 6 and FIGS. 11-18, below.

Neurodegenerative diseases and disorders for which the invention may beeffective include, but are not limited to: Alzheimer's disease,Parkinson's disease, multiple sclerosis, stroke, amyotrophic lateralsclerosis (Lou Gehrig's Disease), frontotemporal dementia (Pick'sDisease), prion disease and Huntington's disease. Additional disordersthat may be treated on the basis of the pharmacological results withestrogens or estrogen receptor active agents, include, but are notlimited to cerebral ischemia, idiopathic Morbus Parkinson, topically- ordrug-induced Parkinson syndrome, Morbus Alzheimer and cerebral dementiasyndromes of different origin, Huntington's chorea, infectious-inducedneurodegeneration disorders such as AIDS-encephalopathy,Creutzfeld-Jakob disease, encephalopathies induced by rubiola and herpesviruses and borrelioses, metabolic-toxic neurodegenerative disorderssuch as hepatic-, alcoholic-, hypoxic-, hypo- orhyperglycemically-induced encephalopathies as well as encephalopathiesinduced by solvents or pharmaceuticals, degenerative retina disorders ofvarious origin, traumatically-induced brain and bone marrow damage,cerebral hyperexcitability symptoms of varying origin such as after theaddition of and/or withdrawal of medicaments, toxins, noxae and drugs,mentally and traumatically-induced cerebral hyperexcitability states,neurodegenerative syndromes of the peripheral nervous system, such asmetabolism, medicament, toxically- and infectiously-inducedpolyneuropathies and polyneuritis, and the bronchospasmolytic effect.

KITS. In another aspect of this invention kits are provided for use bythe treating physician in the clinic or prescribed patient forself-administration of treatment. The kits of this invention include atleast one primary agent and one secondary agent in the appropriatedosages and dosage form for the treatment of the patient's clinicalsymptoms.

In a first embodiment of the kit, the primary agent is estriol in dosesof about 4-16 milligrams and the secondary agent is progesterone indoses of about 100 to about 200 milligrams. In a second embodiment ofthis kit, the primary agent is estriol in doses of about 4-16 milligramsand the secondary agent is a glucocorticoid, such as prednisone (about5-60 milligrams per day) or methyl prednisone (1-2 milligrams per day).

In a third embodiment of this invention, the primary agent is estriol indoses of about 4-16 milligrams and the secondary agent is.beta.-interferon in doses of about 0.25 milligrams of Betaseron® or 30mcg of Avonex® In a fourth alternate embodiment of the kit, the primaryagent is estriol in doses of about 4 to about 16 milligrams and thesecondary agent is glatiramer acetate copolymer in doses of about 20milligrams of Copaxone®

The kit also preferably contains instructions for use of the kit by theuse by the treating physician or patients to treat their autoimmunedisease. Such information would include at least the schedule for theadministration of the primary agent dose and the secondary agent dose.

Although the present invention has been described in terms of thepreferred embodiment above, numerous modifications and/or additions tothe above-described preferred embodiments would be readily apparent toone skilled in the art.

Example 1

Methods: Trial Design.

A crossover design was used with monthly brain MRIs during the six monthpretreatment period, the six month treatment period with oral estriol (8milligrams/day) and the six month post treatment period, with clinicaland laboratory evaluations as demonstrated (FIG. 1A).

Inclusion Criteria.

Women with clinically definite MS, ages 18-50, with an EDSS 0-6.5 whohad been off interferon beta and copolymer-1 for at least six months,and had no steroid treatment for at least three months were eligible. Atleast 5 cm³ of lesion burden on a screening T2 weighted brain MRI wasrequired. Subjects who were pregnant or nursing, on oral contraceptivesor hormone replacement therapy, or who had a history of thrombosis,neoplasm or gynecologic disease, or who had been treated in the pastwith total lymphoid irradiation, monoclonal antibody, T cellvaccination, cladribine or bone marrow transplantation were excluded.

Patients.

Twelve female patients with clinically definite MS were enrolled. Sixhad RR disease and six had SP disease. All six RR and four of six SPpatients completed the entire 18 month study period. One SP patient wasdiscontinued from the study because of prolonged treatment with steroidsfor tonic spasms by an outside neurologist and the other did not wish togo untreated in the post treatment period. Of the ten patients whocompleted the entire study, the mean age was 44 years (range 28 to 50years) and the mean EDSS was 3.3 (range 1.0 to 6.5). The mean EDSS scorefor the SP patients was 5.0 while the mean EDSS for the RR patients was2.2. The 18 month trial was extended in RR patients whereby treatmentwas re-instituted. Medication. For the initial treatment phase,micronized, U.S.P. graded estriol powder (Medisca, Inc., Plattsburg,N.Y.) was put into capsules by UCLA Pharmaceutical Services. During theextension re-treatment phase in the RR patients, all but one received acapsule of estriol (8 milligrams/day) plus progesterone (100milligrams/day), while the single RR patient who had a hysterectomyreceived only estriol (8 milligrams/day) (Women's InternationalPharmacy, Madison, Wis.).

Clinical and Safety Measures.

Subjects were evaluated using the Kurtzke's Expanded Disability StatusScale (EDSS) by the same neurologist (RV) throughout the study. At eachvisit the study nurse (RK) administered the paced auditory serialaddition test (PASAT) and the 9-hole peg test. Blood was drawn forSMA12, cholesterol panel, blood counts and hormone levels (estriol,estradiol, estrone, LH, FSH, cortisol, progesterone). Estriol levels inserum were determined by ELISA according to manufacturer's instructions(Oxford Biomedical, Oxford, Mich.).

Delayed Type Hypersensitivity Responses (DTH).

DTH to tetanus (Tetanus Toxoid, Wyeth Laboratories, Marietta, Pa.) andcandida (Candin, Allermed Laboratories, San Diego, Calif.) were testedat two timepoints, once in the pretreatment period at study month 3 andonce at the end of the treatment period at study month 12 (FIG. 1A). Agroup of six untreated healthy control women were also tested twice,spanning the same time interval (9 months). 0.1 ml of each solution wasinjected intradermally on the anterior surface of the forearm.Induration at each injection site was read after 48 hours. Each site wasmeasured twice, once vertically and once horizontally with the averagerecorded. The same nurse (RK) administered all injections and read allresponses on all subjects at both time points.

Reverse Transcription and Polymerase Chain Reaction.

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized venous blood and cryopreserved. PBMCs were thawed inparallel from a given patient during the two pre-treatment timepointsand the two treatment timepoints. Total RNA was isolated, DNA wasremoved and mRNA was reverse transcribed. Both IFN-.gamma. and actinwere amplified from the same cDNA, however, the cDNA was diluted 1:9prior to amplification for actin. Amplification was done in 1 mMMilligramsCl₂ using IFN.γ and actin primer sequences (Life Technologies,Rockville, Md.). Complementary DNA was amplified for 35 cycles: 45″ @95°C., 60″ @54° C. and 45″ @72° C. PCR products were separated on a 1.5%agarose gel containing ethidium bromide and densitometry performed.

MRIs.

Scans were performed on a 1.5 T G.E. scanner. The pulse sequencesobtained were a T1-weighted scan with and without gadolinium (Omniscan0.1 mmol/kg) and a PD/T2 weighted scan. Digitized image data wastransferred to a SGI workstation (Silicon Graphics, Inc) for furtherprocessing. The number and volume of new and total gadolinium enhancinglesions was determined using a semiautomated threshold based technique(Display, Montreal Neurological Institute) by a single experiencedoperator (NS). The operator was blinded as to whether patients had RR orSP disease. To calculate T2 volumes, a custom semiautomated, thresholdbased, seed-growing algorithm was used to determine lesion volume afterskull stripping, rf correction and spatial normalization. All scans werecounted by the same technician who was blinded as to whether patientshad RR or SP disease.

Statistical Analysis.

One sample, paired, t tests were used to ascertain significance ofpercent changes in DTH responses, IFN.gamma. levels and PASAT cognitivetesting scores during treatment as compared to pretreatment. Thenonparametric, Wilcoxon's signed rank test was used for statisticalcomparisons in enhancing lesion numbers and volumes on MRI between thesix month baseline period and each treatment period, post treatmentperiod and re-treatment period.

Results.

Estriol levels and tolerability. Serum estriol levels during treatmentand re-treatment approximated those observed in women who were sixmonths pregnant, but were lower than those who were 8.5 months pregnant(FIG. 1B). Consistent with previous reports, estriol was well toleratedwith only menstrual cycle abnormalities. There were no significantalterations in any laboratory measures including LH, FSH, cortisol,progesterone, estradiol and estrone.

Immune Responses.

Skin testing to tetanus and-candida were performed once in thepretreatment period and once at the end of the treatment period todetermine whether they might be decreased with treatment. DTH responsesto tetanus were significantly, P=0.006, decreased at study month 12,when patients had been on estriol for six months, as compared to DTHresponses at study month 3, the pretreatment baseline (FIG. 2A). DTHresponses to candida were decreased less dramatically and more variably.The significant decrease in DTH responses to tetanus from pretreatment(month 3) to treatment (month 12) was not merely due to repeat testingat nine months since healthy, untreated female controls tested atbaseline, then again after nine months, did not demonstrate asignificant decrease in DTH responses as compared to their baseline.These findings are consistent with an estriol induced down-regulation ofTh1 responses in vivo during treatment.

IFN.gamma. is a signature cytokine for Th1 responses. Therefore, weassessed IFN.gamma. levels by RT-PCR of unstimulated peripheral bloodmononuclear cells (PBMCs) derived ex vivo from patients during thepretreatment and the treatment periods. In the six RR patients, levelsof IFN.gamma. were variably decreased at study month 9 (after threemonths of estriol treatment) and then significantly decreased, P=0.003,at study month 12 (after six months of estriol treatment) as compared tobaseline pretreatment levels (months 3 and 6) (FIG. 2B). In contrast,there was no decrease in IFN.gamma. in the four SP patients. These dataare consistent with the concept that the immune system of RR patients,as compared to SP patients, may be more amenable to treatments that aimto decrease Th1 responses. Also, the observation that estriol treatmentcan alter cytokine production by PMBCs is consistent with reportsdemonstrating estrogen receptors .alpha. and .beta. in immune tissuesand cells.

MRIs.

Based on the protective effect of pregnancy on relapse rates in MSpatients and the association of gadolinium enhancing lesions withrelapses, we hypothesized that estriol treatment would have ananti-inflammatory effect as manifested by decreases in enhancing lesionson serial brain MRIs. Compared to the six month pretreatment baselineperiod, the total volume and number of enhancing lesions for all ten MSpatients (6RR, 4SP) decreased during the treatment period. Thisimprovement in the group as a whole was driven by the beneficial effectof estriol treatment in the RR, not the SP, group (FIGS. 3A and 3B).Therapeutic effects of estriol treatment in the RR group were thereforeexamined in further detail. Within the first three months of treatmentof RR patients, median total enhancing lesion volumes were decreased by79%, P=0.02, and numbers were decreased by 82%, P=0.09 (FIGS. 3C and3D). They remained decreased during the next three months of treatment,with lesion volumes decreased by 82%, P=0.01, and numbers decreased by82%, P=0.02. In the post treatment period, median total enhancing lesionvolumes and numbers became variable in the first three months offtreatment, before returning to near baseline levels in the last threemonths of the post treatment period. During the four month re-treatmentextension phase, enhancing lesion volumes decreased again by 88%,P=0.008, and numbers decreased again, this time by 48%, P=0.04, ascompared to original baseline (FIGS. 3C and 3D). Changes in median newenhancing lesion volumes and numbers followed similar patterns as mediantotal lesion numbers and volumes (FIGS. 3E and 3F).

Median T2 lesion volumes for the whole group were 15.3 cm.sup.3 (range6.1-33.8), with no significant differences in median T2 volumes betweenRR and SP groups. Consistent with enhancing lesion data, serial T2lesion volumes revealed that estriol treatment tended to be mostbeneficial in RR patients. In the RR group, median T2 lesion volumesremained stable during the six month treatment period (0% change),increased during the six month post treatment period (7.4% higher), andthen declined in the four month re-treatment extension period (2.0%lower).

Clinical Measures.

Relapses were few and showed no significant changes during the study. Inthe six RR patients, one relapse occurred during the pretreatmentperiod, one in the treatment period, two in the post treatment periodand none in the re-treatment period. No relapses occurred in SPpatients. EDSS and 9 Hole Peg Test scores showed no significant changesduring the study (Table 1).

TABLE I Clinical Measures EDSS scores Pretreatment Estriol TreatmentPost Treatment 3 mo. 6 mo. 9 mo. 12 mo. 15 mo. 18 mo. 6 RR 2.2 2.0 1.51.7 1.8 1.8 (0.6) (0.5) (0.7) (0.6) (0.6) (0.5) 4 SP 5.0 5.0 4.9 5.0 5.15.0 (0.9) (0.9) (1.0) (0.9) (1.1) (0.8) 9 Hole Peg Test scoresPretreatment Estriol Treatment Post Treatment 3 mo. 6 mo. 9 mo. 12 mo.15 mo. 18 mo. 6 RR R 22.2 21.8 22.5 21.5 21.0 21.4 (2.4) (1.6) (2.3)(1.9) (1.7) (2.4) L 24.8 22.9 24.3 23.3 23.0 22.7 (3.2) (1.6) (2.5)(2.1) (2.1) (2.3) 4 SP R 26.8 29.9 30.2 31.7 29.4 34.0 (0.4) (2.4) (1.4)(4.8) (5.2) (8.7) L 23.5 25.6 22.7 24.8 26.7 25.0 (1.4) (2.5) (1.7)(2.6) (0.7) (1.8)Interestingly, PASAT cognitive testing scores were significantlyimproved in the RR-group, but not in the SP group (FIG. 4). Thisimprovement in PASAT scores in RR patients by 14.0% during treatment ascompared to baseline, reached statistical significance, P=0.04. It isunlikely that this improvement was entirely due to a practice effect ofrepeated testing because of the long time interval between testing (9months) and because alternate versions of the test were used in eachpatient. This beneficial effect of estriol treatment on PASAT scores ofRR MS patients is consistent with previous reports describing abeneficial effect of estrogen replacement therapy in surgicallymenopausal women and high dose estrogen treatment in Alzheimer'sdisease. Sicottte, et al. Treatment of Women with Multiple SclerosisUsing Pregnancy Hormone Estradiol: A Pilot Study. Neurology, 56 (8 Supp.3):A75, April 2001, and Sicottte, et al. Treatment of Multiple Sclerosiswith the Pregnancy Hormone Estradiol, Submitted to Neurology 2002, areherein incorporated by reference in their entirety.

Example 2

Progesterone in combination with estrogen treatments has been shown toprotect against endometrial proliferation and cancer. Indeed, estrogencannot be given for a lengthy period of time in an “unopposed” fashionin any woman with a uterus. Thus, seven of the 12 patients wanted toremain on estriol after completion of the 18 month study. These patientswere then put back on 8 milligrams of estriol and 100 milligrams ofprogesterone per day. In an extension phase of the study which beganafter completion of the post treatment phase. This extension phase was 4months in duration. Each of the seven patients had an MRI every monthduring the 4 month extension phase. Additionally, each of the sevenpatients was examined neurologically and had serologic studies done atthe end of this phase. No known negative effects 100 milligrams ofprogesterone in combination therapy with 8 milligrams of estrioltreatment were noted.

Example 3

In a pilot clinical trial, non-pregnant female MS patients were treatedwith estriol to induce a pregnancy level in serum. This treatmentreduced the prototypic in vivo Th response, the delayed typehypersensitivity response, as well as reduced Th1 (TNFα, IFNγ) andincreased TH2 (IL5, IL10) cytokine production by peripheral bloodmonuclear cells (Siotte et al., 2002; Soldan et al., 2003). Also,gadolinium-enhancing lesions on serial brain magnetic resonance images(MRIs) were reduced by >80% (Sicotte et al., 2002). Because enhancinglesion activity on brain MRI is a putative biomarker for relapses in MS,these reports together suggested that estriol treatment may recapitulatethe anti-inflammatory effect of pregnancy in relapsing remitting MS(RRMS).

Example 4

A 33 year old white female patient was diagnosed as having relapsingremitting multiple sclerosis. Following the delivery of her first child(now age 7), the patient was treated only with Copaxone and relapsed at6 weeks. Following the delivery of her second child (now age 3), thepatient was again treated with Copaxone alone and again relapsed, thistime at 4.5 months. Following a subsequent pregnancy, the patient wastreated with 8 mg estriol/day in an attempt to prevent her post partumrelapses.

Following the birth of the patient's third child (now 6 months), thepatient resumed treatment with Copaxone as before. However, on day 10post-partum she began taking estriol 8 mg/daily in an oral dosage form.The patient had no relapses for 6 months post-partum, and her neurologicexam is unchanged with minimal disability (EDSS=1). Since monthly brainMRIs with gadolinium to detect enhancing MS lesions are more sensitivefor inflammatory disease activity than relapses, the patient underwentserial monthly MRIs at post partum months 4, 5, and 6. There was noenhancement at month 4, only one small enhancing lesion at month 5, andat 6 months only a small residual, less robust enhancement of the singlelesion from the previous month. No new enhancement was observed at month6. The T2 lesion load has been stable throughout.

The patient has had increased irregular menstrual bleeding despite usingthe progesterone minipill (norethindrone, 0.35 mg daily), one pill perday since day 10, to stabilize the uterine endometrium and for birthcontrol. Uterine ultrasounds at month 3 and 6 showed a thin, not thick,endometrium, consistent with an unstable lining, not suggestive ofhyperplasia. The patient doubled the progesterone minipill for 2 weeksto stabilize the endometrium. Otherwise no adverse events have beenreported.

Example 5

Animals. Female C57BL/6 mice, 8 weeks of age, were purchased fromTaconic (Germantown, N.Y.). ERα KO mice backcrossed onto the C57BL/6background for 16 generations were a generous gift from Dr. DennisLubahn (University of Missouri, Columbia, Mo.) (Lubahn et al., 1993).Wild-type littermates from F16 crosses served as ERα KO matchedcontrols. ERβ KO mice, a generous gift from Dr. Jan Ake Gustafsson(Karolinska Institute, Stockholm, Sweden) (Krege et al., 1998), werebackcrossed onto the C576BL/6 background for eight generations.Wild-type littermates from these crosses served as ERβ KO matchedcontrols. Animals were housed under guidelines set by the NationalInstitutes of Health, and experiments were conducted in accordance withthe University of California, Los Angeles Chancellor's Animal ResearchCommittee and the Public Health Service Policy on Humane Care and Use ofLaboratory Animals.

Reagents.

PPT was purchased from Tocris Bioscience (Ellisville, Mo.), and E2 waspurchased from Sigma-Aldrich (St. Louis, Mo.). Miglyol 812 N, a thinliquid oil, was obtained from Sasol North America (Houston, Tex.).Myelin oligodendrocyte glycoprotein (MOG) peptide, amino acids 35-55,was synthesized to >98% purity by Chiron Mimotopes (San Diego, Calif.).

EAE.

Active EAE induction ensued with subcutaneous injection of an emulsioncontaining the autoantigen MOG peptide, amino acids 33-55 (300 μg/mouse)and Myobacterium tuberculosis (500 μg/mouse) in complete Freund'sadjuvant, as described previously (Suen et al., 1997; Liu et al., 2003).Mice underwent hormonal treatments as described below and were monitoreddaily for EAE disease severity using the standard EAE grading scale, asdescribed previously (Pettinelli and McFarlin, 1981). Briefly, todetermine the clinical score for each mouse on each day, each mouse wasgraded using the standard 0-5 scale: 0, unaffected; 1, tail limpness; 2,failure to right on attempt to roll over; 3, partial paralysis; 4,complete paralysis; and 5, moribund. On each day, the mean of theclinical scores of all mice within a given treatment group weredetermined, thereby yielding the mean clinical score for that treatmentgroup. Some mice were followed clinically for up to 40 d after diseaseinduction, and others were killed earlier for mechanistic studies, 1-2 dafter the onset of clinical signs in the vehicle-treated group (day16-19 after disease induction).

Treatments.

Isoflurane-anesthetized female mice were ovariectomized and allowed torecuperate for 10 days. Daily treatments of oil vehicle alone,estradiol, or PPT began 7 days before EAE immunization. Estradiol andPPT were dissolved in 10% ethanol and 90% oil to give the final properconcentration of 0.04 mg/kg/day of estradiol (Jansson et al., 1994) and10 mg/kg/d of PPT per mouse (Harris et al., 2002). Estradiol, PPT orvehicle alone were given by daily subcutaneous injections along themidbackline and continued for the entire disease duration (up to 40 daysafter disease induction).

Perfusion.

Mice were deeply anesthetized with isoflurane and perfusedtranscardially with ice-cold 0.9% saline, followed by 10% formalin.Spinal cord columns were removed and postfixed overnight in 10% formalinand cryoprotected with 20% sucrose solution, in PBS. Spinal cords wereremoved from the column, cut in three parts (cervical, thoracic, andlumbar), and embedded in gelatin/sucrose mix. Spinal cord regions ingelatin were further postfixed and stored in 20% sucrose. Free-floatingsections (25 μm thick) were cut coronally with a sliding microtome andcollected serially in PBS.

Uterine Weights.

After the mice were killed, each uterus was extracted, and the fat,connective tissue, and excess fluid were removed to obtain each uterineweight, as described previously (Frasor et al., 2003).

Immune Responses.

Spleens were harvested during deep anesthesia before perfusion.Splenocytes were stimulated with the autoantigen, MOG peptide 35-55, at25 μg/ml. Supernatants were collected after 48 and 72 h, and levels ofTNFα, interferon-γ (IFNγ) interleukin-6 (IL6), and IL5 were determinedby cytometic bead array (BD Biosciences Pharmingen, San Diego, Calif.)as described previously (Liu et al., 2003).

Histopathology and Immunohistochemistry.

Serial sections ere mounted on slides and stained with hematoxylin andeosin (H&E), Niss1, or Luxol fast blue (LFB)-cresyl violet. Consecutivesections were also examined by immunohistochemistry. Briefly, 25 μmfree-floating sections were permeabillized in 0.3% Triton X-100 in PBSand blocked with 10% normal goat serum. White matter immunostaining wasenhanced by treating sections with 95% ethanol/5% acetic acid for 15 minbefore permeabilization and blocking. To detect specific cell types andstructures, sections were preincubated with primary antibodies in PBSsolution containing 2% NGS for 2 h at room temperature, and thenovernight at 40° C. The following primary antibodies were used: anti-β3tubulin and anti-neurofilament-NF200 [monoclonal (Chemicon, Temecula,Calif.); polyclonal (Sigma Biochemical)], anti-neuronal-specific nuclearprotein (NeuN), anti-CD45 (Chemicon), anti-myelin basic proteins (MBP;Chemicon) and anti-Mac 3 (BD Biosciences Pharmingen). The secondantibody step was performed by labeling with antibodies conjugated toTRITC, FITC, and Cy5 (Vector Laboratories and Chemicon). IgG controlexperiments were performed for all primary antibodies, and no stainingwas observed under these conditions. To assess the number of cells, anuclear stairs 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; 2ng/ml; Invitrogen, Eugene, Oreg.) was added for 15 min before finalwashes after secondary antibody addition. The sections were mounted onslides, dried, and coverslipped in fluoromount G (Fisher Scientific,Hampton, N.H.).

Microscopy.

Stained sections were examined and photographed using a confocalmicroscope (TCS-SP; Leica, Mannheim, Germany) or a fluorescencemicroscope (BX51WI; Olympus, Tokyo, Japan) equipped with Plan Fluorobjectives connected to a camera (DP70; Olympus). Digital images werecollected and analyzed using Leica confocal and DP70 camera software.Images were assembled using Adobe Photoshop (Adobe Systems, San lose,CA).

Quantification.

To quantify immunostaining results, sections from spinal cord levelsT1-T5 were examined, six from each mouse, with n=3 mice per treatmentgroup, for a total of 18 sections per treatment group. Images werecaptured under microscope (4×, 10×, or 40×) using the DP70 Imagesoftware and a DP70 camera (both from Olympus). Identical lightintensity and exposure times were applied to all photographs from eachexperimental set. Images from the same areas of spinal cord werecompared (T1-T5) and were acquired separately from delineated whole grayand white matter regions. The middle region of the ventral horn was thefocus for gray matter analysis, whereas the area lateral to the ventralhorn was the focus for white matter analysis. Six gray matter and sixwhite matter pictures were collected from the two sides of T1-T5sections (100 μm apart) from three animals in each treatment group. Allimages were converted to grayscale and then analyzed by densitymeasurement with ImageJ version 1.29 (the Windows version of NIH Image),downloaded from rsb.info.nih.gov/ij. A fixed threshold range of 0-160was chosen to highlight the staining signals in normal spinal cordsections, and the total area within this range was measured, averaged,and compared.

Increase in total number of infiltrating cells after induction of EAEwas measured by density measurements of DAPI+ nuclei in the whole whitematter. Neuronal cells were quantified by counting theNeuN+/β3-tubulin+/DAPI+ cells per square millimeter in the whole graymatter. Both white and gray matter assessments occurred its the T1-T5spinal cord sections. Laser-scanning confocal microscopic scans at 40×were performed on Mac 3+β/3-tubulin+ immunostained spinal cord sectionscorresponding to levels T1-T5 ventral horn. The results for eachexperimental condition were averaged from four unilateral levels permouse (100 μm apart, three mice in each treatment group, total of 12sections per treatment group) and were expressed as mean fold changecompared with healthy match controls.

Statistical Analysis.

EAE disease severity was compared between groups using the Friedmantest, histopathological changes were assessed using 1×4 ANOVAs, anduterine weights and cytokine levels were compared between treatmentgroups using Student's t test, as described previously (Dalal et al.,1997).

Results.

Treatment with an ERα ligand remains highly selective for ERα in vivoduring EAE.

The dose of the ERα-selective ligand for use in our RAE experimentswhich could induce a known biological response on a control tissue (theuterus). Estrogen-induced increases in uterine weight had been shownpreviously to be mediated by ERα, and doses of the ERα ligand PPT neededfor this in vivo treatment effect had been described (Frasor et al.,2003). Daily subcutaneous injections of PPT, at a dose previously shownto increase uterine weight (10 mg/kg/d), resulted in a significantincrease in uterine weight in female C57BL/6 mice with EAE at day 40after disease induction, FIG. 1A. Sensitivity of this technique wasshown by the decrease in uterine weight in ovariectomized compared withsham-operated, vehicle-treated mice. Treatment with injections of highdoses of estradiol (to induce pregnancy levels in serum) served as apositive control, whereas treatment with injections of vehicle aloneserved as a negative control. To further demonstrate the in vivoselectivity of this dose of PPT, uterotrophic responses were alsoexamined during PPT treatment of ERα or ERβ knock-out mice. Significantincreases in uterine weight were observed in PPT-treated ERβ knock-outmice (FIG. 1B) but not in ERα knock-out mice (FIG. 1C). Together, thesedata demonstrated that the method of administration of the ERα ligandPPT induced an expected biological response in vivo on a positivecontrol tissue.

FIG. 5 depicts results showing results showing treatment with anERα-selective ligand is highly selective in vivo during EAE. As shown inFIG. 5A, treatment with the ERα ligand PPT induced expected biologicalresponses on uterine weight (y-axis=uterine weight in grams). Uterineweight was increased with PPT given as daily subcutaneous injections at10 mg/kg/day. The decrease in uterine weight with ovariectomy comparedwith sham surgery demonstrated the sensitivity of the technique indetecting differences in uterine weights associated with differences inestrogen levels. Treatment with a dose of estradiol known to induce alate pregnancy level of estradiol was used ad a positive control for anincrease in uterine weight, whereas treatment with vehicle alone servedas the negative control. The uteri were removed at day 35-40 during EAEtreatment with the indicated hormone (sham vehicle, n=6; OVX vehicle,n=12; OVX estradiol, n=18; OVX PPT, n=18). OVX PPT and OVX Estradiol,each as compared with OVX Vehicle, ***p<0.0001. WT<Wild type. As shownin FIG. 5B, Uterine weights were examined in ovariectomized ERβknock-out mice as in FIG. 5A. Uterine weights were increased with PPTtreatment in ERβ knock-out mice (OVX vehicle, n=9; OVX estradiol, n=12;OVX PPT, n=12). OVX PPT and OVX Estradiol, each as compared with OVXVehicle, *** p<0.0001. As shown in FIG. 5C, Uterine weights wereexamined in ovariectomized ERα knock-out mice as in FIG. 5A. Uterineweights were not increased with PPT treatment in ERα knock-out mice (OVXvehicle, n=6; OVX estradiol, n=4; OVX PPT, n=6).

Treatment with an ERα ligand reduces the clinical severity of EAE. Usingthe above dose and method of administration, PPT treatment was assessedfor its effect on the clinical course of EAE. Ovariectomized, C57BL/6wild-type female mice with MOG 35-55 peptide-induced active EAE weretreated with the ERα-selective ligand PPT. PPT treatment significantlyreduced the clinical severity of EAE (FIG. 6A). Treatment withinjections of estradiol served as a positive control, whereas treatmentwith injections of vehicle alone served as the negative control.

When ovariectomized ERβ knock-out C57BL/6 female mice were treated withPPT-during active EAE, clinical disease severity was also significantlydecreased (FIG. 6). These data demonstrated that the presence of ERβ wasnot required for disease protection mediated by treatment with PPT. Incontrast, when PPT was administered to ovariectomized ERα knock-out miceinduced with active EAE, the disease-ameliorating effect of PPTtreatment was abolished, as evidenced by the lack of a difference inmean clinical scores when comparing PPT-treated and vehicle-treated ERαknock-out mice (FIG. 6C). Similar results were obtained when castratedmale mice were used instead of ovariectomized females (data not shown),consistent with a previous publication demonstrating thatestrogen-medicated improvements in clinical EAE in castrated male micewere abrogated in the ERα knock-out (Liu et al., 2003). ERα knock-outfemale mice have high circulating estradiol levels; hence, estrogenunresponsiveness in this mouse could be attributable to the ERα geneticmodification or the estrogen history of the mouse before ovariectomy at4 weeks. Because male ERα knock-out mice do not have high circulatinglevels of estradiol, similar results in both the female and male ERαknock-outs make the ERα genetic modifications, not the estrogen historyof the mouse, most likely responsible for effects observed.

These data demonstrated that the estrogen-medicated protection from EAEcould be recapitulated by treatment with a highly selective ERα ligand,and that this protection was not dependent on an interaction with ERβ.

FIG. 6. Treatment with an ERα-selective ligand is sufficient to reducethe clinical severity of EAE. As shown in FIG. 6A, EAE clinical severitywas decreased in ovariectomized, wild-type (WT) C57BL/6 female micetreated with PPT. Daily treatments of ovariectomized mice withinjections of vehicle (negative control), estradiol (positive control),or PPT (10 mg/kg/day) began, and then 7 d later, active EAE was inducedwith MOG 35-55 peptide. Mean clinical scores were significantly reducedin both estradiol- and PPT-treated mice compared with vehicle treated(p<0.0001, Friedman test). Data are representative from experimentsrepeated a total of five times. As shown in FIG. 6B, the decrease in themean clinical scores of EAE by PPT treatment was not dependent on thepresence of ERβ. Ovariectomized, ERβ knock-out C57BL/6 female mice weretreated with either PPT, estradiol, or vehicle as in A. Mean clinicalscores were significantly reduced in both estradiol- and PPT-treatedmice compared with vehicle treated (p<0.0001, Friedman test). Data arerepresentative from experiments repeated a total of three times. Asshown in FIG. 6C, PPT treatment in vivo during EAE remains highlyselective for ERα. Ovariectomized female ERα knock-out C57BL/6 mice weretreated as in FIG. 6A. In ERα knock-out mice, mean clinical scores werenot significantly different in PPT-treated compared withvehicle-treated. PPT-treated wild-type mice served as a positive controlfor a PPT treatment effect within the experiment. Data arerepresentative from experiments repeated a total of three times. Errorbars indicate variability of clinical scores between mice within a giventreatment group. n=5 mice per each treatment group.

Treatment with an ERα Ligand Reduces Autoantigen-SpecificProinflammatory Cytokine Production.

Because it had been shown previously using ERα knock-out mice that bothdisease protection and a reduction in proinflammatory cytokines (TNFαand IFNγ) were dependent on ERα, we next determined whether treatmentwith an ERα ligand could reduce proinflammatory cytokine production. Asdemonstrated in FIG. 7, PPT treatment significantly reduce TNFα, IFNγ,and IL6 production. Interestingly, we had shown previously thatproduction of the Th2 cytokine IL5 was increased with estrogen treatmentand that this was only partially, but not completely, abolished in theERα knock-out (Liu et al., 2003). In the present study, when wild-typemice were treated with the ERα agonist PPT, treatment significantlyincreased IL5 production. Together, these data demonstrated thattreatment with an ERα agonist induced changes in cytokine productionduring autoantigen-specific immune responses in the peripheral immunesystem that would be anti-inflammatory with respect to EAEimmunopathogenesis.

As shown in FIG. 7, treatment Treatment with an ERα ligand reducedproinflammatory cytokine production by peripheral immune cells inovariectomized, wild-type C57BL/6 female mice with EAE. EAE was inducedas in FIG. 6, and then at day 40 after disease induction, mice werekilled, and cytokine production by MOG 35-55 stimulated splenocytes wasdetermined. TNFα, IFNγ, and IL6 levels were each significantly reducedwith PPT treatment, whereas IL5 levels were increased with PPTtreatment. Error bars indicate variability of cytokine values forsplenocytes between individual mice within a given treatment group, withn=5 mice for each treatment group. Data are representative ofexperiments repeated three times. *p<0.05.

Treatment with an ERα Ligand Reduces Inflammation and Demyelination inEAE.

Because we had observed that treatment with the ERα ligand PPTrecapitulated the protective effect of estrogen treatment on theclinical course of EAE and was anti-inflammatory with respect to theautoantigen-specific immune response in the periphery, we nextascertained the effect of treatment with PPT on inflammation anddemyelination in the CNS of EAE mice. Spinal cord sections ofovariectomized, C57BL/6 mice at the acute phase of EAE (1-2 days afteronset of clinical signs in vehicle-treated mice) were assessed forinflammation and demyelination. Mice from all treatment groups werekilled at the same time point, to permit their examination in parallel.Compared with vehicle-treated EAE, both inflammation and demyelinationwere markedly reduced by treatment with the ERα ligand PPT or E2 (FIG.4). H&E-stained vehicle-treated EAE mice, compared with normal healthycontrols, had numerous multifocal to coalescing inflammatory cellinfiltrates in the spinal cord. Infiltrates were present in theleptomeninges, around blood vessels in the leptomeninges, and in theparenchyma of the white matter (FIG. 4A). Inflammatory cell infiltrateswere associated with pallor and vacuolation, consistent withdemyelination. Quantification of white matter cell density by countingDAPI+ cells revealed a 60% increase in infiltrates of vehicle-treatedEAE group. In contrast, both estradiol and PPT treated mice had nodetectable inflammation, with white matter cell densities similar tothose in the normal control (FIG. 4D).

The degree of myelin loss was assed by Luxol fast blue and confirmed byMBP immunostaining. Luxol fast blue staining revealed demyelination atthe sites of inflammatory cell infiltrates (FIG. 4B). Also, myelinstaining of dorsal column regions of vehicle-treated spinal cord sectionhad significantly less MBP immunostaining compared with normal control,E2-, and PPT-treated sections, FIG. 4C. Quantification of demyelinationby density analysis of Luxol fast blue-stained spinal cord sectionsrevealed a 25% decrease in myelin density in vehicle-treated EAE mice.In contrast, both estradiol- and PPT-treated mice had much lessdemyelination, with myelin densities not significantly different fromthose in the normal control (FIG. 8E).

As shown in FIG. 8, treatment with an ERα ligand reduced inflammationand demyelination in spinal cords of mice with EAE. In FIG. 8A,representative H&E-stained thoracic spinal cord sections (4×magnification) from normal (healthy control), as well as vehicle-, E2-,and PPT-treated EAE mice are shown. Vehicle-treated EAE mouse spinalcord shows multifocal to coalescing areas of inflammation in theleptomeninges and white matter, around blood vessels, and in theparenchyma of the white matter (areas of inflammation shown by arrows).No inflammation was observed in either E2- or PPT-treated EAE spinalcords. As shown in FIG. 8B, luxol fast blue-stained region of dorsalcolumn (square in A) of spinal cords (40× magnification). Intensedemyelination in the white matter is seen in vehicle-treated EAEsections only. As shown in FIG. 8C, anti-MBP immunostained dorsal columndemonstrated demyelination in the white matter of vehicle-treated EAEsections only. As shown in FIG. 8D, increase in total number ofinfiltrating cells after induction of EAE was semiquantified by countingDAPI+ cells in the entire delineated white matter (including dorsal,lateral, and ventral funiculi) and presented as percentage of normal.Vehicle-treated EAE mice had a significant increase in white matter celldensity compared with healthy normal control, whereas E2-treated and theERα ligand (PPT)-treated groups did not. As shown in FIG. 8E, the extentof demyelination was compared by staining thoracic spinal cord sectionswith Luxol fast blue. Myelin density is presented as percentage ofnormal. Vehicle-treated mice EAE mice had a significant decrease inmyelin density in the entire delineated white matter as compared withnormal control, whereas E2-treated and PPT-treated groups did not.Number of mice, three per treatment group; number of T1-T5 sections permouse, six; total number of sections per treatment group, 18.**Statistically significant compared with normals (p<0.001), 1×4 ANOVAs.Data are representative of experiments repeated in their entirety onanother set of EAE mice with each of the treatments.

Treatment with an ERα Ligand is Neuroprotective in EAE.

In light of the significant anti-inflammatory effect induced by PPTtreatment of mice with EAE, the preservation of neuronal and axonalintegrity was examined. A combination of Niss1 stain histology andanti-NeuN/β3-tubulin immunolabeling was used to identify andsemiquantify neurons, and neurofilament antibody (anti-NF200) was usedto identify axons. At the acute phase of EAE, 1-2 d after the onset ofclinical signs in vehicle-treated mice, thoracic spinal cord sections ofall treatment groups of EAE mice were assessed for NeuN+/β3 tubulin+neurons in the gray matter and NF200 axons in the white matter. Asurprising decrease in neuronal staining (NeuN+/Niss1+) in gray matteroccurred at this early time point in vehicle-treated EAE mice (FIG. 9B)compared with normal, healthy, age- and gender-matched control mice(FIG. 9B). This significant decrease in neuronal staining in gray matterof vehicle-treated EAE mice was not observed in EAE mice treated witheither estradiol (FIG. 9C) or the ERα ligand (FIG. 9D). Quantificationof NeuN⁺ cells in gray matter confirmed the significant loss invehicle-treated EAE mice compared with normal controls, whereasestradiol- and PPT-treated mice had NeuN+ cell numbers that were nodifferent from the normal control (FIG. 9E).

As shown in FIG. 9, treatment with an ERα ligand preserved neuronalstaining in gray matter of spinal cords of mice with EAE. As shown inFIGS. 9A-D, split images of thoracic spinal cord sections stained withNeuN (red) in i and Niss1 in ii at 4× magnification, derived from normalhealthy control mice (A), vehicle-treated EAE (B), E2-treated EAE mice(C), and ERα ligand (PPT)-treated EAE mice (D), each killed very earlyduring EAE, 1-2 days after the onset of clinical signs. iii, Mergedconfocal scan at 40× of NeuN⁺ (red) and β3-tubulin+ (green) colabeledneurons from an area represented by dotted white square area in i. iv, A40× magnification of Niss1-stained area in solid black square in ii. Adecrease in NeuN⁺ immunostaining and Niss1 staining was observed in thedorsal horn, intermediate zone, and ventral horn of vehicle-treated EAEmice (FIG. 9B) compared with normal controls (FIG. 9A). White arrows inBiii denote loss of NeuN⁺ staining. In contrast, EAE mice treated witheither estradiol (FIG. 9C) or PPT (FIG. 9D) had preserved NeuN⁺ andNiss1 staining. After quantification of neurons in the entire delineatedgray matter of T1-T5 sections, NeuN⁺ immunolabeled neurons weresignificantly decreased, by nearly 25%, in vehicle-treated EAE micecompared with normal controls, but E2- and PPT-treated EAE mice were notstatistically different from normal controls (FIG. 9E). Number of mice,three per treatment group; number of T1-T5 sections per mouse, six;total number of sections per treatment group, 18. **Statisticallysignificant compared with normals (p<0.001); 1×4 ANOVAs. Data arerepresentative of experiments repeated in their entirety on another setof EAE mice with each of the treatments.

Immunostaining for neurofilament (NF200) resulted in clearidentification of axons within the spinal cord of normal mice (FIG.10A). A significant decrease in axonal NF200 staining (NF200+) in whitematter occurred in vehicle-treated EAE mice compared with normalcontrols in areas positive for CD45 staining, consistent with previousobservations of axonal transection within inflammatory white matterlesions in EAE (Wujek et al., 2002). EAE mice treated with eitherestradiol or the ERα ligand demonstrated not decrease in axonal NF200+staining and only an occasional single cell positive for CD 45 (FIG.10A). Quantification of axon numbers in white matter confirmed thesignificant loss in vehicle-treated EAE mice, but no significant axonalloss occurred in EAE mice treated with either estradiol or the ERαligand (FIG. 10C). These immunohistological data are consistent with ourobservation of markedly reduced inflammatory lesions by H&E in whitematter with these treatments (FIG. 8A). Notably, at this early timepoint in EAE, there was no loss in axon numbers in white matter areasdevoid of inflammatory lesions, even in the vehicle-treated EAE group,thereby providing no evidence for Wallerian degeneration of white mattertracts in these regions of the cord at this very early time point inEAE.

Treatment with an ERα Ligand Reduces Microglial/Monocyte Activation inWhite and Gray Matter of Mice with EAE.

Gray matter axonal pathology has been described in cortex of MSpatients, which was characterized by activated microglia closely opposedto and ensheathing apical dendrites, neuritis, and neuronal perikarya(Peterson et al., 2001). In light of our observation of a decrease inNeuN⁺/β3-tubulin⁺/Niss1⁺ neuronal staining in the gray matter of spinalcords in EAE, we next addressed the microglial reaction in this graymatter. Microglia/monocytes were stained for Mac 3, a lysosomal antigenequivalent to LAMP-2 (lysosomal-associated membrane protein 2)/CD107b,present on the surface of microglia and mature mononuclear phagocytes,and sections were coimmunolabeled with anti-B3-tubulin (FIG. 10B).Striking Mac 3+ reactivity was observed in gray matter of mice at thisvery early time point in EAE, only 1-2 days after the onset of clinicalsigns in the vehicle-treated group. Most of the MAC 3+ cellsdemonstrated a morphology similar to that of activated microglia (FIG.10B, inset). They were in close vicinity to, and in direct contact with,gray matter neurons that had reduced and punctuate β3-tubulin staining(FIG. 10B). In contrast, EAE mice treated with either the ERα ligandPPT, or estradiol, which were killed and examined in parallel, had some,but significantly less, immunoreactivity (FIG. 10B). Quantification ofMAC 3+ cells revealed an ˜65% decrease when E2- and PPT-treated spinalcords were compared with those from vehicle-treated EAE mice (FIG. 10D).

As shown in FIG. 10, treatment with an ERα ligand reduced CD45+ and Mac3+ cells in white and gray matter of mice with EAE. As shown in FIG.10A, thoracic spinal cord sections from mice used in FIG. 9 werecoimmunostained with NF200 (green) and CD45 (red) at 10× magnification.Shown are partial images with white and gray matter from normal control,vehicle-treated EAE, E2-treated EAE, or ERα ligand (PPT)-treated EAEmice. LF, Lateral funiculus of white matter; GM, gray matter. Thevehicle-treated EAE cords had large areas of CD45+ cells associated withreduced NF200 axonal staining in white matter compared with the normalcontrol, whereas estradiol and ERα ligand-treated EAE mice had onlyoccasional CD45 positivity, with intact NF200 axonal staining. As shownin FIG. 10B, consecutive sections from the same mice were alsocoimmunostained with β3-tubulin (green) and Mac 3 (red), with thesection of the ventral horn designated by the dotted line square area inFIG. 10A scanned at 40× magnification by confocal microscopy.Vehicle-treated EAE mice demonstrated markedly increased Mac 3 stainingin ventral horn gray matter compared with normal control mice, with mostof these Mac 3+ cells having the morphology of microglia (inset, 100×magnification). They were surrounding neuronal structures (whitearrows). In contrast, E2- and ERα ligand (PPT)-treated EAE cord sectionsdemonstrated less Mac 3 immunostaining compared with vehicle-treated EAEmice. As shown in FIG. 10C, after quantification, neurofilament-stainedaxon numbers in white matter were significantly lower in vehicle-treatedEAE mice compared with normal mice, whereas E2- and PPT-treated EAE micedemonstrated no significant reduction in axon numbers. Axon number ispresented as percentage of normal. **Statistically significant comparedwith normal (p<0.001); 1×4 ANOVAs. FIG. 10D, Mac 3× cells were analyzedby density measurements and represented as percentage of vehicle-treatedgroups. Compared with vehicle-treated EAE mice, both the E2-treated andPPT-treated had significantly lower Mac 3+ immunoreactivity in graymatter. Number of mice, three per treatment group; number of T1-T5sections per mouse, four; total number of sections per treatment group,12. **Statistically significant compared with normal (p<0.001); 1×4ANOVAs. Data are representative of experiments repeated in theirentirety on another set of EAE mice with each of the treatments.

Example 6 The Neuroprotective Effects of Estrogen Receptor (ER) Beta

Methods. Animals. Female wild type C57BL/6 mice, as well as female ERJ31(0 mice on the C57BL16 background, age 8 weeks, were obtained from‘laconic (Germantown, N.Y.). Wild type SIIL female mice, age S weeks,were obtained from Harlan laboratories (Indianapolis, Ind.). Animalswere maintained in accordance with guidelines set by the NationalInstitutes of Health and as mandated by the University of California LosAngeles Office for the Protection of Research Subjects and theChancellor's Animal Research Committee.

Reagents.

Propyl pyrazole triol (PPt and Diarylpropionitrile (DPN), an ERα and anERβ agonist, respectively, were purchased from Tocris Bioscience(Ellisville, Mo.). Estradiol was purchased from Sigma-Aldrich (St.Louis, Mo.). Miglyol 812 N, a thin liquid oil, was obtained from SasolNorth America (Houston, Tex.). Myelin oligodendrocytes glycoprotein(MOO) peptide, amino acids 35-55, proteolipid protein (PLP) peptides139-151 and 179-191, and myelin basic protein (MBP) peptide 83-102 weresynthesized to >98% purity by Mimotopes (Clayton, Victoria, Australia).

Uterine Weights to Assess Dosing.

Uterine weight was used as a positive control to assess dosing ofestrogen agonists. Daily subcutaneous injections of vehicle, estradiol,PPT, or DPN, as well as a combination of ITT with DPN, were administeredfor ten days at indicated doses to ovariectomized mice. Followingeuthanasia, the uterus was extracted, then fat, connective tissue, andexcess fluid removed in order to obtain the uterine weight, asdescribed.

Hormone Manipulations During EAE.

Isotlurane-anesthetized female mice were ovaricctornized and allowed torecuperate for 7-10 days. Daily subcutaneous injections of vehicle,estradiol, PPT, or DPN began seven days prior to EAE immunization, andcontinued throughout the entire disease duration. Estradiol wasdelivered at a concentration of 0.04 mg/kg/day, DPN at 8 mg/kg/day andITT at 10 mg/kg/day. Vehicle alone treatments consisted of 10% Ethanoland 90% Migylol.

EAE Induction.

Active EkE was induced by immunizing with 300 gg of myelinoligodenrocyte glycoprotein (MOO) peptide, amino acids 35-55, and 500 pgof Mycobacterium tuberculosis in complete Freund's adjuvant asdescribed. Active EAE was induced in SiT, mice with 100 jig ofproteolipid protein (PLP) peptide, amino acids 139-151, and 100 jig ofMycobacterium tuberculosis in complete Freund's adjuvant as described.Mice were monitored and scored daily for clinical disease severityaccording to the standard 0-5 EAE grading scale: 0, unaffected; 1, taillimpness; 2, failure to right upon attempt to roll over; 3, partialparalysis; 4, complete paralysis; and 5, moribund. On each day, the meanof the clinical scores of all mice within a given treatment group weredetermined, thereby yielding the mean clinical score for that treatmentgroup. Some mice were followed clinically for up to 50 days afterdisease induction, while others were sacrificed earlier for mechanisticstudies at day 19 after disease induction, corresponding to day 4-6after the onset of clinical signs in the vehicle treated group.

Rotarod Testing.

Motor behavior was tested up to two times per week for each mouse usinga rotarod apparatus (Med Associates mc, St. Albans, Vt.). Briefly,animals were placed on a rotating horizontal cylinder for a maximum of200 seconds. The amount of time the mouse remained walking on thecylinder, without falling, was recorded. Each mouse was tested on aspeed of 3-30 rpm and given three trials for any given day. The threetrials were averaged to report a single value for an individual mouse,and then averages were calculated for all animals within a giventreatment group. The first two trial days, prior to immunization (day0), served as practice trials.

Immune Responses.

Spleens were harvested either after deep anesthesia prior to perfusionor after euthanasia. Splenocytes were stimulated with the indicatedautoantigens at 25 pg/mI, and proliferation assessed using standard H3incorporation assays, as described. Supernatants were collected after 48and 72 hours, and levels of TNF-i, IFN-γ, 11,6, and 1L5 were determinedby cytometric bead array (BD Biosciences), as described.

Perfusion.

Mice were deeply anesthetized with isoflurane and perfusedtranscardially with ice-cold 0.9% saline, followed by 10% formalin.Spinal cord columns were removed and post-fixed overnight in 10%formalin and cryoprotected with 20% sucrose solution in PBS. Spinalcords were removed from the column and cut in 3 parts (cervical,thoracic and lumbar) and embedded in gelatin/sucrose mix. Spinal cordregions in gelatin were further postfixed and stored in 20% sucrose.Free-floating sections (25 pm thick) were cut coronally with a slidingmicrotome and collected serially in PBS.

Histopathology and Immunohistochemistry.

Serial sections were mounted on slides and stained with Hematoxylin &eosin (H&E) or Niss1. Consecutive sections were also examined byimmunohistochemistry. Briefly, 25 μm free-floating sections werepermeabilized in 0.3% Triton X-100 in PBS and blocked with 10% normalgoat serum. White matter immunostaining was enhanced by treatingsections with 95% ethanol/5% acetic acid for 15 minutes prior topermeabilization and blocking. To detect specific cell types andstructures, sections were pre-incubated with primary antibodies in PBSsolution containing 2% NGS for 2 hours at room temperature, thenovernight at 40 C. The following primary antibodies were used: anti-β3tubulin and anti-neurofilament-NF200 (monoclonal, Chemicon; polyclonalSigma Biochemical), anti-neuronal specific nuclear protein (NeuN),anti-CD4S (Chemicon), and anti-MW (Chemicon). The second antibody stepwas performed by labeling with antibodies conjugated to TRITC, FITC andCy5 (Vector Labs and Chemicon). IgG-control experiments were performedfor all primary antibodies, and no staining was observed under theseconditions. To assess the number of cells, a nuclear stain4′,6-Diamidino-2-phenylindole, DAPI (2 ng/ml; Molecular Probes) wasadded for 15 minutes prior to final washes after secondary antibodyaddition. The sections were mounted on slides, dried and coverslipped influoromount G (Fisher Scientific).

Microscopy.

Stained sections were examined and photographed using a confocalmicroscope (Leica TCS-SP, Mannheim, Germany) or a fluorescencemicroscope (BX51WI; Olympus, Tokyo, Japan) equipped with Plan Fluorobjectives connected to a camera (DP70, Olympus). Digital images werecollected and analyzed using Leica confocal and DP70 camera software.Images were assembled using Adobe Photoshop (Adobe Systems, San Jose,Calif.).

Quantification.

To quantify immunostaining results, sections from spinal cord levelsT1-T5 were examined, six from each mouse, with n=3 mice per treatmentgroup, for a total of 18 sections per treatment group. Images werecaptured under microscope (4×, 10× or 40×) using the DP70 Image softwareand a DP70 camera (both from Olympus). Identical tight intensity andexposure times were applied to all photographs from each experimentalset. Images from the same areas of spinal cord were compared (TI-IS) andwere acquired separately from delineated whole gray and white mailerregions. The middle region of the ventral horn was the focus for graymatter analysis, while the area lateral to the ventral horn was thefocus for white matter analysis. Six gray matter and six white matterpictures were collected from the two sides of TI-IS sections (100 jimapart) from three animals in each treatment group. All images wereconverted to grayscale and then analyzed by density measurement withImageJ vi 0.29 (the Windows version of NIH Image), downloaded fromrsb.info.nih.gov/ij. A fixed threshold range of 0 to 160 was chosen tohighlight the staining signals in normal spinal cord sections, and thetotal area within this range was measured, averaged, and compared.

Increase in total number of infiltrating cells after induction of EAEwas measured by density measurements of DAPI⁺ nuclei in the whole whitematter. Neuronal cells were quantified by counting theNeuN⁺/β3-tubulin+/DAPI⁺ cells per mm2 in the whole gray matter. Bothwhite and gray matter assessments occurred in the TI-IS spinal cordsections. Laser scanning confocal microscopic scans at 40× wereperformed on Mac 3+/β3-tubulin⁺ immunostained spinal cord sectionscorresponding to levels 11-15 ventral horn. The results for eachexperimental condition were averaged from four unilateral levels permouse (100 pm apart, three mice in each treatment group, total of 12sections per treatment group) and were expressed as mean fold change ascompared to healthy matched controls, as described.

Statistical Analysis.

EAE clinical disease severity was compared between treatment groupsusing the Friedman test; histopathological changes were assessed using1×4 ANOVAs; uterine weights, proliferative responses and cytokine levelswere compared between treatment groups using Student t-test, and time onrotorod was compared between treatment groups using ANOVA.

Results.

Selected doses of ERα and ERβ ligands induced known biological responseson a positive control tissue, the uterus. Before beginning EAEexperiments, the uterine response was used to assess whether a known invivo response would occur during treatment with each of our dosingregimens. It was known that estrogen treatment increased uterine weightprimarily though ERα, and it had also been shown that treatment with theERβ ligand Diarylpropionitrile (DPN) could antagonize the ERα mediatedincrease in uterine weight. The ERα ligand propyl pyrazole triol (PPT)was given to ovariectomized C7BL/6 females for 10 days at either anoptimal (10 mg/kg/day) or suboptimal (3.3 mg/kg/day) dose, and asignificant increase in uterine weight as compared to vehicle treatedwas observed (FIG. 11). For the ERβ ligand DPN, a dose was selectedwhich was shown to be neuroprotective in an animal model of globalischemia. When this DPN dose (8 mg/kg/day) was given in combination withPPT treatment, the increase in uterine weight mediated by PPT treatmentwas significantly reduced. Doses of the ERα and ERβ ligands induce knownbiological responses on a positive control tissue. C57BLI6 mice wereovariectomized, then treated for 10 days with indicated doses of ERα orERβ ligands as daily subcutaneous injections to determine the effect ofthis dosing regimen on uterine weight. As shown in FIG. 11, uterineweight was increased with PPT treatments at both 10 mg/kg/day and 3.3mg/kg/day, as compared to vehicle treated controls. Treatment with DPNalone at 8 mg/kg/day had no effect on uterine weight, while this DPNdose antagonized the PPT 3.3 mg/kg/day mediated increase in uterineweight. Each treatment group, n=4. * indicates p<0.05, student t-test.

These data demonstrated that the method and dose of delivery of the ERαand ERβ ligands induced a known biological response in vivo on apositive control tissue, the uterus.

Differential Effects of Treatment with ERα and ERβ Ligands on ClinicalEAE.

We compared and contrasted effects between ERα and ERβ treatment duringEAE. When the ERα ligand was administered one week prior to active EAEinduction with MOG 35-55 peptide in ovariectomized C57BL/6 female mice,clinical disease as measured by the standard EAE grading scale wascompletely abrogated, p<0.0001 (FIG. 12A). This was consistent with ourpreviously findings in this EAE model (described above), as well asfindings in adoptive EAE in SJL mice by others. In contrast, ERβ ligandtreatment had no significant effect early in disease (up to day 20 afterdisease induction), but then demonstrated a significant protectiveeffect later in disease (after day 20), p<0.001 (FIG. 12B).

The protective effect using the ERβ ligand DPN in active EAE in C57BL/6mice were surprising given that another ERβ ligand (WAY-202041) wasshown to have no effect in adoptive EAE in SJL mice. Since WAY-202041was shown to have a 200 fold selectivity for ERβ as compared to ERα,while DPN has a 70 fold selectivity, it was possible that DPN was notsufficiently selective for ERβ in vivo in our studies. To assess the invivo selectivity of DPN during EAE, DPN was administered to ERβ KO mice.When OPN was administered to ovariectomized ERβ KO C57BL/6 mice withactive EAE, the treatment was no longer protective (FIG. 12C). Thesedata demonstrated the in vivo selectivity of DPN for ERβ during EAE atthe dose used.

Together these results indicate that treatment with an ERα ligand isprotective throughout the course of EAE, while treatment with an ERβligand is protective during the later phase of the disease, alter theacute initial phase.

Differential Effects of Treatment with ERα and ERβ Ligands onAutoantigen Specific Cytokine Production in C57BL/6 Mice with EAE.

To further investigate differences between treatments with the ERαversus the ERβ ligand, the autoantigen specific cytokine productionduring both early and later stages of EAE in C57BL/6 mice was assessed.ERα ligand treatment significantly reduced levels of proinflammatorycytokines (TNFα, IFNγ, and IL6), while increasing the anti-inflammatorycytokine IL5, during both early (FIG. 12D) and later (FIG. 12F) stagesof EAE. In contrast, ERβ ligand treatment was not statisticallydifferent from vehicle treatment in all measured cytokines (TNFα, IFNγ,and IL6, and IL5) at either the early (FIG. 12E) or later (FIG. 12G)time points. Treatment with ERα versus ERβ selective ligands hasdifferential effects on chronic EAE and autoantigen specific immuneresponses in C57BL/6 mice. Ovariectomized C57BL/6 female mice were givendaily subcutaneous injections of an ER ligand during active EAE andgraded using the standard EAE grading scale. FIG. 12A, Mean clinicalscores of PPT treated mice as compared to vehicle treated mice weresignificantly reduced during the entire disease course, p<0.0001,Friedman test. Each treatment group had an n=4, and data arerepresentative of a total of five repeated experiments. FIG. 12B, DPNtreated mice, as compared to vehicle treated mice, were notsignificantly different early in disease (up to day 20 after diseaseinduction), but then became significantly improved later during EAE,(following day 30 after disease induction) p<0.001, Friedman test.Number of mice in each group were vehicle, n=4; estradiol, n=4; DPN,n=8. Data are representative of experiments repeated twice. DPNtreatment in vivo during EAE remains highly selective for ERβ. Clinicalscores in ovariectomized ERβ 1(0 C57BL/6 mice with active EAE were nodifferent when comparing DPN treated with vehicle treated. Eachtreatment group had an n 4, and data are representative of experimentsrepeated twice. Estradiol treated mice served as a positive control fora treatment effect in each experiment (FIGS. 12A-C).

At day 19 (FIGS. 12D and 12E) or day 40 (FIGS. 12F and 12G) afterdisease induction, mice were sacrificed and cytokine production by MOO35-55 stintulated splenocytes was determined. ITT treatmentsignificantly reduced TNFα, LFNγ, and LL6, and increased LU during earlyEAE (FIG. 12D) and late EAE (FIG. 12F).

In contrast, no significant differences with DPN treatment were seen inmeasured cytokine levels at either the early stage (FIG. 12E) or latestage (1) of EAE disease. Error bars indicate variability of cytokinevalues for individual mice within a given treatment group, with n=4 micefor each treatment group. Data are representative of two to fiveexperiments for each time point. (FIGS. 12D-G) No differences wereobserved with either ERα or ERβ ligand treatment, as compared tovehicle, for IL1O production, while 11,4 and 1L12 levels were too low todetect (not shown).

These results indicated that while ERα ligand treatment inducedfavorable changes in cytokine production during the autoantigen specificimmune response, ERβ ligand treatment did not.

Treatment with an ERβ Ligand Reduces Clinical Relapses, but does notAlter Autoantigen Specific Immune Responses in SJL Mice with EAE.

Next, proteolipid protein (PLP) 139-151 induced active EAE in SJL micewere treated with either DPN or vehicle control. While there was nodifference in the incidence, the day of onset, or the peak clinicalscores, there was a significant decrease in relapses in DPN treated mice(5/13, 33%) as compared to vehicle treated (10/13, 77%), p<0.01. Theserelapses occurred between days 36 and 52 after disease induction.Notably, the previous report stating that the ERβ ligand WAY-202041 wasnot protective in EAE in SJL mice followed mice for only the first 27days after disease induction, a duration including only the firstepisode of acute EAE, and a time when no effect of DPN treatment wasobserved.

The immune responses in this EAE model were then assessed. Since epitopespreading had been previously described in SJL mice with PLP 139-151induced EAE, the immune response to the disease initiating autoantigen(PLP 139-151) was assessed, as well as the response to possible epitopespreading autoantigens (PLP 179-191 and MBP 83-102). There was nosignificant effect of ERβ legand treatment, as compared to vehicletreatment, on immune responses to the disease initiating autoantigen(FIGS. 13A-C), and no epitope spreading occurred, even m vehicle treatedEAE mice, consistent with some reports not detecting epitope spreading.

FIGS. 13A-C. Treatment with an ERβ selective ligand did not affectperipheral immune cells in SJL mice with EAE. Active EAE was inducedwith PLP 139-151 peptide in ovariectomized SJL female mice treated witheither vehicle, DPN or estradiol. At day 52 after disease induction,mice were sacrificed and splenic immune responses to the diseaseinitiating antigen (PLP 139-151), as well as to possible epitopespeading antigens (PLP 178-191 and MBP 83-102) were assessed. The onlydetectable response in all three treatment groups was to the diseaseinitiating antigen (PLP 139-151), while responses to possible epitopespeading antigens were undetectable. No significant differences wereobserved in proliferation or cytokine (TNFα or LENγ) production duringthe PLP 139-151 specific response in the DPN treated group as comparedto the vehicle treated group. Estradiol treatment served as the positivecontrol for a treatment effect on immune responses, demonstratingdecreases in the proliferative response, as well as in TNFα and IFNγcytokine production, when compared to vehicle treated, consistent withprevious reports. Error bars indicate variability of values forindividual mice within a given treatment group, with n=4 mice for eachtreatment group, and data are representative of experiments repeatedtwice.

Together these data indicated that while ERβ ligand treatment mediated areduction in relapses in SW mice with EAE, the mechanism for this effecton relapses did not include a significant effect on cytokine productionor epitope spreading.

Treatment with an ERα Ligand, but not an ERβ Ligand, Reduces CNSInflammation in EAE.

The comparison of the effect of ERα versus ERβ ligands in neuropathologywas assessed. At both early (day 19) and later (day 40) stages of EKE,spinal cord sections from mice treated with either vehicle, ERα or ERβligand were assessed for inflammation and demyelination. On hemotoxylinand eosin (H&E) staining, vehicle treated C578L16 EAE mice had extensivewhite matter inflammation at both the early (FIG. 14A) and later (FIG.14C) time points as compared to the healthy controls. As compared tovehicle treated EAE, this inflammation was significantly reduced bytreatment with the ERα ligand PPT. In contrast, extensive white matterinflammation was present in the ERβ ligand treated group at both theearly and late timepoints. Quantification of white matter cell densityby counting DAPI+ cells revealed that ERα ligand treated mice at theearly stage of EAE had a significant, p<0.001, reduction in inflammationin white matter of the thoracic cord as compared with vehicle treatedEAE, while white matter cell densities in DPN treated EAE mice were notsignificantly different from those in vehicle treated, FIG. 14B. At thelater time point, quantification revealed a lesser, but stillsignificant, p<0.05, reduction in inflammation with ERα ligand treatmentas compared to vehicle, while inflammation in ERβ ligand treated was nodifferent from that in vehicle treated, FIG. 14D.

Double immunohistochemistry using anti-CD4S and anti-NIF200 antibodieswas then used to stain inflammatory cells and axons, respectively. ERαligand treated EAE mice, as compared to vehicle treated EAE, had lessC045 staining in white matter. This reduction in C045 staining was mostmarked at the early time point in EAE (FIG. 14E), while at the latertime point, some C045 staining was detectable in the ERα ligand treated,albeit still less than in vehicle treated (FIG. 14F). In contrast, ERβligand treated EAE mice did not have reduced CD45 staining in whitematter, at either the early or the later time points.

Additionally, CD45 staining of cells in gray matter of vehicle treatedEAE mice was observed at both the early and later time points, and thesecells had a morphology suggestive of activated microglia (FIGS. 14E andF insets), ERα ligand treatment, but not ERβ ligand treatment, reducedthis CD4S staining in gray matter.

Together these data indicated that ERα ligand treatment, but not ERβligand treatment, reduced inflammation in the CNS of mice with EAE.Notably, the lack of a reduction in CNS inflammation with ERβ ligandtreatment was consistent with the lack of an immunomodulatory effect ofERβ ligand treatment on the autoantigen specific immune response in theperiphery (FIG. 12).

Treatment with Both an ERα Ligand and an ERβ Ligand ReducesDemyelination and Axonal Transaction in White Matter in EAE.

The degree of myelin loss was then assessed by myelin basic protein(MBP) immunostaining in the dorsal columns of thoracic cords. Extensivedemyelination occurred at the sites of inflammatory cell infiltrates invehicle treated EAE mice while less demyelination occurred in ERα andERβ ligand treated (FIGS. 15A and 15C). Quantification of demyelinationby density analysis of MBP immunostained spinal cord sections revealed a32% (p<0.01) and 34% (p<0.005) decrease in myelin density in vehicletreated EAE mice, at the early and later time points, respectively, ascompared to normal controls (FIGS. 17B and 17D). Myelin staining wasrelatively preserved in both ERα and ERβ ligand treated mice, at boththe early and later time points in disease, with reductions ranging from7-19%, not significantly different than healthy controls.

Staining with anti-NF200 antibody revealed axonal loss in white matterof vehicle treated mice at both early and later time points of diseaseas compared to normal controls, while both ERα ligand and ERβ ligandtreatment resulted in less axonal loss, as compared to that in vehicletreated EAE mice (FIGS. 15E and 15G). Quantification of NF200 stainingin anterior fununculus revealed a 49±12% (p<0.01) and 40±8% (p<0.005)reduction in vehicle treated EAE, at the early and later time points,respectively, as compared to healthy controls (FIGS. 15F and 15H) Axonnumbers in ERα ligand and ERβ ligand treated EAE mice were notsignificantly reduced as compared to those in healthy controls.

FIG. 15. Treatment with an ERα ligand and an ERβ ligand each preservedmyelin basic protein immunoreactivity and spared axonal pathology inwhite matter of spinal cords of mice with EAE. Dorsal columns ofthoracic spinal cord sections were imaged at lox magnification from micein FIG. 14 that were immunostained with antiMBP (red). At day 19 (FIG.15A) and day 40 (FIG. 15C) after disease induction, vehicle treated micehad reduced MBP immunoreactivity as compared to normal controls, whilePPT treated EAE and DPN treated EAE mice showed relatively preserved MBPstaining. Upon quantification (FIGS. 15B and 15D), MBP immunoreactivityin dorsal column was significantly lower in vehicle treated EAE mice ascompared to normal mice, while PPT and DPN treated EAE mice demonstratedno significant decreases. Myelin density is presented as percent ofnormal. Statistically significant compared with normal (*p<0.01;p<0.005), 1×4 ANOVAs.

Part of the anterior funniculus of thoracic spinal cord sections wasimaged at 40× magnification from mice in FIG. 15 that wereco-immunostained with anti-NF200 (green, i) and anti-MBP (red, ii).Merged images of smaller (i) and (ii) panels are shown in (iii).Distinct green axonal centers surrounded by red myelin sheaths can beseen in normal controls, PPT and DPN treated EAE mice from 19 day (FIG.15E) and 40 day (FIG. 15G) after disease induction. Vehicle treated miceshow reduced axonal numbers and myelin, along with focal demyelination(white stars) and loss of axons. Upon quantification (FIGS. 15F and15H), neurofilament stained axon numbers in white matter weresignificantly lower in vehicle treated EAE mice as compared to normalmice, while PPT and DPN treated EAE mice demonstrated no significantreduction in axon numbers. Axon number is presented as percent ofnormal. Statistically significant compared with normal (*p<0.01;**p<0.005), 1×4 ANOVAs.

Together these data demonstrated that ERα ligand treatment reducedinflammation, demyelination and axonal transection in white matterduring EAE, while ERβ ligand treatment did not reduce inflammation, butnevertheless still was capable of reducing demyelination and axonaltransection.

Treatment with both an ERα ligand and an ERβ ligand reduces neuronalpathology in gray mailer of mice with EAE.

In Example 5 above, we demonstrated neuronal abnormalities surprisinglyearly during EAE (day 15), which were prevented by treatment with eitherestradiol or PPT. Whether ERβ ligand treatment might preserve neuronalintegrity at both the early (day 19) and later (day 40) time points ofEAE was examined. Using a combination of Niss1 stain histology and antiNeuN/β3-tubulin immunolabeling of neurons in gray matter were identifiedand quantified, at both the early and later time points in EAE. Adecrease in neuronal staining in gray matter occurred at both timepoints in vehicle treated EAE mice as compared to normal controls, whileneuronal staining in gray matter was well preserved in EAE mice treatedwith either the ERα or the ERβ ligand at the early and the later timepoints (FIGS. 16A and 16C). Quantification of NeuN⁺ cells in gray matterdemonstrated a 41±13% (p<0.05) and 31±8% (p<0.05) reduction, at theearly and later time points respectively, in vehicle treated EAE mice ascompared to normal controls, while PPT and DPN treated mice had NeuN⁺cell numbers that were fewer, but not significantly different from thosein healthy controls (FIGS. 16B and 16D).

FIG. 16. Treatment with an ERα ligand and an ERβ ligand each preservedneuronal staining in gray matter of spinal cords of mice with EAE. Splitimages of thoracic spinal cord sections stained with NeuN⁺ (red) in (i)and Niss1 in (ii) at 4× magnification, derived from normal healthycontrol mice, vehicle treated EAE, ERα ligand (PPT) treated EAE and ERβligand (DPN) treated EAE mice, each sacrificed at either day 19 (early;FIG. 16A) or at day 40 (late; FIG. 16C) after disease induction. Panel(iii) is a merged confocal scan at 40× of NeuN⁺ (red) and(33-tubulin+(green) co-labeled neurons from an area represented bydotted white square area in (i). Panel (iv) is a 40× magnification ofNiss1 stained area in solid black square in (ii). A decrease in NeuN⁺immunostaining and Niss1 staining was observed in the dorsal horn,intermediate zone and ventral horn of vehicle treated EAE mice ascompared to normal control. White arrows in panel (iii) denote loss ofNeuN⁺ staining. In contrast, EAE mice treated with either PPT or DPN hadpreserved NeuN and Niss1 staining. Upon quantification of neurons in theentire delineated gray matter of T1-T5 sections, NeuN⁺ immunolabeledneurons were significantly decreased, by nearly 41%, in vehicle treatedEAE mice at day 19 (FIG. 16B) and nearly 31% at day 40 (FIG. 16D) ascompared to normal controls, while PPT and DPN treated EAE mice were notstatistically different from normal controls. Number of mice 3 pertreatment group, number of T1-T5 sections per mouse=6, total number ofsections per treatment group=18. Statistically significant compared withnormals (*p<0.05), 1×4 ANOVAs. Data are representative of experimentsrepeated in their entirety on another set of EAE mice with each of thetreatments.

Protection from neuropathology is mediated by ERβ.

To confirm whether the effect of DPN treatment in vivo on CNSneuropathology was indeed mediated through ERβ, we next assessed whiteand gray matter neuropathology in DPN treated EAE mice deficient in ERβ.At day 38 after disease induction, inflammation, demyelination andreductions in axon numbers were present in white matter, while neuronalstaining was decreased in gray matter of vehicle treated EAE mice (FIG.17). In contrast to the preservation of myelin, axon numbers andneuronal staining observed during DPN treatment of wild type mice (FIGS.15 and 16), DPN treatment of ERβ knock out mice failed to prevent thiswhite and gray matter pathology (FIG. 17).

FIG. 17. DPN treatment mediated protection from neuropathology duringEAE is dependent upon ERβ. As shown in FIG. 17A, part of the anteriorfunniculus of thoracic spinal cord sections from ERβ knock out controlmice, vehicle treated EEβ knock out with EAE and DPN treated ERβ knockout with EAE at day 40 after disease induction were imaged at 40×magnification upon co-immunostaining with anti-NF200 (green, i) andanti-MBP (red, ii). Merged images are shown in panel iii. ERβ knock outcontrol sections showed robust NF200 and MBP immunostaining similar towild type normal controls in FIG. 18, whereas vehicle and DPN treatedEAE sections had decreased myelin and axonal staining. FIG. 17B showssplit images of thoracic spinal cord sections, derived from mice in FIG.17A, stained with NeuN (red) in (i) and Niss1 in (ii) at 4×magnification, showed neuronal losses in gray matter of both the vehicletreated and DPN treated ERβ knock out mice with EAE. (FIGS. 17C-F)Quantification of white matter cell density, myelin density, axonalnumbers and NeuN⁺ cells revealed that DPN treatment does not preventwhite and gray matter pathology during EAE in ERβ knock out mice. Numberof mice=3 per treatment group, number of T1-T5 sections per mouse=6,total number of sections per treatment group=18. Statisticallysignificant compared with normals (**p<0.001), 1×4 ANOVAs. These datademonstrate that direct neuroprotective effects mediated by DPNtreatment in vivo during EAE are mediated through ERβ.

Treatment with an ERβ Ligand Induces Recovery of Motor Performance.

Since treatment with an ERβ ligand was found to be neuroprotective inEAE, the clinical significance of this neuroprotective effect wasassessed. The clinical outcome frequently used in spinal cord injury,rotarod performance was used. Vehicle treated C57BL/6 EAE micedemonstrated an abrupt and consistent decrease in the number of secondsthey were able to remain on the rotarod, beginning at day 12 afterdisease induction (FIG. 18A). This disability remained throughout theremainder of the observation period in vehicle treated EAE mice. Incontrast, ERβ ligand treated mice had an abrupt decrease in the numberof seconds they could remain on the rotarod apparatus, beginning at day12, but later during EAE, at days 30-40, they had significant recoveryof their ability to remain on the rotarod. These data demonstrated thatERβ ligand treatment induces functional clinical recovery in motorperformance at later time points of disease during EAE.

Finally, to assess whether the improvement in rotarod performance withDPN treatment was mediated through ERβ, rotarod performance studies wereconducted in ERβ KO female mice. The improvement in rotarod performancelate during EKE with DPN treatment was no longer observed in the ERβ KO(FIG. 18B).

FIG. 18. Treatment with an ERβ selective ligand results in recovery ofmotor function late during EAE. Ovariectoniized C57BL/6 female mice withEAE were treated with DPN and assessed for motor performance on arotarod apparatus. As shown in FIG. 18A, while mean time on rotaroddecreased abruptly at day 12 after disease induction in both the vehicleand DPN treated EAE mice, after day 30 the DPN treated groupdemonstrated significant recovery of motor function, while the vehicletreated did not improve. *p<0.01 and ** p<0.005, ANOVA. Estradioltreatment served as a positive control for a treatment effect. Number ofmice in each treatment group, vehicle n=4; DPN n=8; estradiol n=4. Dataare representative of experiments repeated twice. As shown in FIG. 18B,in contrast to the improvement observed with DPN treatment of wild typemice, no improvement was observed at the later phase of disease in DPNtreated ERβ KO mice. Again, vehicle served as a negative control, andestradiol served as a positive control, for a treatment effect. Numberof mice in each treatment group, vehicle n=4; DPN n=4; estradiol n=4.

These data demonstrated that the DPN induced recovery in motorperformance later in disease was mediated through ERβ.

In closing, it is noted that specific illustrative embodiments of theinvention have been disclosed herein above. However, it is to beunderstood that the invention is not limited to these specificembodiments.

Accordingly, the invention is not limited to the precise embodimentsdescribed in detail hereinabove. With respect to the claims, it isapplicant's intention that the claims not be interpreted in accordancewith the sixth paragraph of 35 U.S.C. Section 112 unless the term“means” is used followed by a functional statement.

While the specification describes particular embodiments of the presentinvention, those of ordinary skill can devise variations of the presentinvention without departing from the inventive concept.

1-47. (canceled)
 48. A method of treating a secondary progressive formof multiple sclerosis comprising administering to a patient apharmaceutical composition comprising estriol at a therapeuticallyeffective dosage in an effective dosage form.
 49. The method of claim 48wherein the therapeutically effective dosage is about 0.001 to about 16milligrams about every 24 hours.
 50. The method of claim 48 wherein thetreatment results in patient serum concentrations of estriol of about 2to about 30 nanograms per milliliter.
 51. The method of claim 48 furthercomprising administering to the patient progesterone in an effectivedosage in an effective dosage form.
 52. The method of claim 51 whereinthe progesterone is administered in a dosage of 100 milligrams per day.53. A method of treating a secondary progressive form of multiplesclerosis comprising administering to a patient an oral pharmaceuticalcomposition comprising estriol at a dosage of about 8 milligrams aboutevery 24 hours.
 54. The method of claim 53 wherein the treatment resultsin patient serum concentrations of estriol of about 2 to about 30nanograms per milliliter.
 55. The method of claim 53 further comprisingadministering to the patient progesterone in an effective dosage in aneffective dosage form.
 56. The method of claim 53 wherein theprogesterone is administered in a dosage of 100 milligrams per day.